Acetyl coa carboxylase 2 sequences and methods

ABSTRACT

The present invention relates generally to novel nucleotide and amino acid sequences, and more particularly to novel human acetyl CoA carboxylase 2 (ACC2) and rat ACC2 sequences. The sequences provided herein can be expressed in a recombinant format. Methods of isolating the ACC2 sequence are also provided, which can be employed to isolate any ACC sequence. The ACC2 sequences can be employed in therapeutic applications to diagnose or treat a condition associated with ACC2. The invention also relates to the identification of modulators of ACC activity using the recombinant human ACC2 enzyme as the screening target.

This application is a division of U.S. application Ser. No. 11/186,999,filed Jul. 21, 2005, which claims priority benefit to U.S. provisionalapplication No. 60/590,948 filed Jul. 23, 2004. The entire teachings ofthe referenced application are incorporated herein by reference.

FIELD OF THE INVENTION

Disclosed and claimed herein are novel polynucleotides encoding humanand rat Acetyl CoA Carboxylase 2 (“ACC2”) polypeptides, fragments andhomologues thereof. Vectors, host cells, antibodies, and recombinant andsynthetic methods for producing the ACC2 polypeptides are provided.Further described are diagnostic and therapeutic methods for applyingthe ACC2 polypeptides to the diagnosis, treatment, and/or prevention ofvarious diseases and/or disorders related to these polypeptides,including obesity. Further embodiments include screening methods foridentifying agonists and antagonists of the polynucleotides andpolypeptides of the present invention.

BACKGROUND OF THE INVENTION

Acetyl CoA carboxylase (ACC) is the rate-determining enzyme of fattyacid biosynthesis in plants and animals. ACC is a biotin containingenzyme which catalyzes the carboxylation of acetyl CoA to form malonylCoA in a two-step reaction (Beaty & Lane, (1982). J. Biol. Chem.257:924-929). The first step is the ATP-dependent carboxylation ofbiotin covalently linked to the enzyme. In the second step, acarboxyltransferase step, the carboxyl group is transferred to thesubstrate, acetyl CoA, to form malonyl CoA. Citrate is a potentallosteric activator of ACC. Malonyl CoA is the C2 donor for de novosynthesis of long chain fatty acids.

In mammals, there are two subtypes of ACC, ACC1 and ACC2. ACC1 is mainlylocalized in lipogenic tissues such as adipose tissue and liver, wherefatty acids are synthesized. ACC2 is found primarily in non-lipogenictissues such as skeletal muscle and heart muscle, although some is alsofound in liver. Malonyl CoA allosterically inhibits carnitine palmitoyltransferase 1 (CPT1), which is a critical enzyme to transfer the longchain fatty acid into the mitochondria for β-oxidation. Because ACC2 isco-localized with CPT-1, the primary role of malonyl CoA that issynthesized by ACC2 has been suggested to regulate the rate ofβ-oxidation.

ACC is a potential target in metabolic diseases for the treatment ofmetabolic syndrome including obesity, insulin resistance anddyslipidemia. Increased rates of muscle fatty acid oxidation, a reducedfat content and a reduction in total body fat were observed in ACC-2knock-out mice (Abu-Elheiga et al., (2001) Science 291:2613-2616;Abu-Elheiga et al., (2003) Proc. Natl. Acad. Sci. USA. 100:10207-10212).Harwood et al. reported that ACC inhibitors caused reduction in fattyacid synthesis, increase in fatty acid oxidation, and reduction ofrespiratory quotient in rats (Harwood et al., (2003) J. Biol. Chem.278:37099-37111). Chronic dosing of these compounds resulted in thereduction of whole body fat mass and improvement of insulin sensitivity(Harwood et al., (2003) J. Biol. Chem. 278:37099-37111). Theseobservations further validated the enzyme as a drug target.

Several human ACC2 and rat ACC2 nucleotide and amino acid sequences havebeen published (see, e.g., Human ACC2: GenBank Accession No.NM_(—)001093 (SEQ ID NOs:1 and 2) and GenBank Accession No. AC007637(SEQ ID NOs:3 and 4); Rat ACC2: GenBank Accession No. NM_(—)053922 (SEQID NOs:7 and 8) and GenBank Accession No. AB004329 (SEQ ID NOs:9 and10)). It was found, however, that for each species, each of thepublished amino acid and/or nucleotide sequences was different from oneanother by one or more residues. More specifically, it was found thatthe nucleotide sequences of human ACC2 and rat ACC2 contain non-silentmutations that introduce substitutions into several of the publishedencoded amino acid sequences of these enzymes.

In order to identify the most effective modulators of human ACC2 and ratACC2, accurate nucleotide and amino acid sequences are required.Therefore, what is needed to advance research on human and rat ACC2 isan accurate amino acid sequence for these enzymes, as well as theencoding nucleotide sequences. The subject matter disclosed and claimedherein solves this and other problems.

SUMMARY OF THE INVENTION

Described and claimed herein is an isolated nucleic acid moleculeencoding a human ACC2 polypeptide. In one embodiment the nucleic acidmolecule comprises a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a polynucleotide encoding anACC2 polypeptide comprising SEQ ID NO:12; (b) an isolated polynucleotideencoding a human ACC2 polypeptide comprising amino acids 2 to 2458 ofSEQ ID NO:12 minus the start methionine; (c) an isolated polynucleotideencoding a human ACC2 polypeptide comprising amino acids 1 to 2458 ofSEQ ID NO:12 including the start codon; (d) an isolated polynucleotideencoding the ACC2 polypeptide encoded by the cDNA clone contained inATCC Deposit No: PTA-6054; and (e) a polynucleotide capable ofhybridizing under stringent conditions to the polynucleotide specifiedin (a)-(d), wherein the polynucleotide does not hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence of only A residues or of only T residues. The isolated nucleicacid molecule can comprise, for example, the nucleotide sequence of SEQID NO:11. In additional aspects, the present invention also relates to apolynucleotide that is complementary to the isolated nucleic acidmolecule, a vector comprising the isolated nucleic acid molecule and ahost cell, which can be a mammalian host cell, comprising the vector.

Also disclosed is a method of making an isolated human ACC2 polypeptide.In one embodiment the method comprises: (a) culturing the recombinanthost cell under conditions such that the polypeptide is expressed; and(b) recovering the polypeptide.

Further described is an isolated human ACC2 polypeptide. In oneembodiment the polypeptide comprises an amino acid sequence selectedfrom the group consisting of: (a) a polypeptide comprising SEQ ID NO:12;(b) a polypeptide comprising amino acids 2 to 2458 of SEQ ID NO:12,wherein amino acids 2 to 2458 comprise a polypeptide of SEQ ID NO:12minus the start methionine; and (c) a polypeptide comprising amino acids1 to 2458 of SEQ ID NO:12. In another embodiment, the polypeptidecomprises two or more sequential amino acid deletions from one or bothof: (a) the COOH-terminus of the polypeptide; and (b) the NH₂-terminusof the polypeptide.

Another embodiment disclosed herein is a method of identifying acompound that modulates the activity of a human ACC2 polypeptide. In oneembodiment, the method comprises: (a) determining the activity of anACC2 polypeptide in the absence of a test compound; (b) determining theactivity of the polypeptide in the presence of a test compound; and (c)comparing the activity of the polypeptide in the presence of the testcompound with the activity of the polypeptide in the absence of the testcompound, wherein a change in the activity of the polypeptide in thepresence of the test compound relative to the activity of thepolypeptide in the absence of the test compound indicates that thecompound that modulates the activity of the polypeptide.

A further embodiment is an isolated antibody which specifically binds toa human ACC2 polypeptide. In various embodiments, the antibody isselected from the group consisting of a chimeric antibody, a singlechain antibody, a Fab fragment, and a humanized antibody.

Further disclosed and claimed is an isolated nucleic acid moleculeencoding a rat ACC2 polypeptide. In one embodiment the isolated nucleicacid molecule comprises a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a polynucleotide encoding anACC2 polypeptide comprising SEQ ID NO:14; (b) an isolated polynucleotideencoding a rat ACC2 polypeptide comprising amino acids 2 to 2458 of SEQID NO:14 minus the start methionine; (c) an isolated polynucleotideencoding a rat ACC2 polypeptide comprising amino acids 1 to 2458 of SEQID NO:14 including the start codon; (d) the cDNA of ATCC Deposit No.PTA-6054; and (e) a polynucleotide capable of hybridizing understringent conditions to the polynucleotide specified in (a)-(d), whereinthe polynucleotide does not hybridize under stringent conditions to anucleic acid molecule having a nucleotide sequence of only A residues orof only T residues.

In one embodiment, the isolated rat nucleic acid molecule comprises thenucleotide sequence of SEQ ID NO:13. In additional aspects, the presentinvention comprises a polynucleotide that is complementary to theisolated nucleic acid molecule, a vector comprising the isolated nucleicacid molecule and a host cell, which can be a mammalian host cell,comprising the vector.

Another embodiment relates to a method of making an isolated rat ACC2polypeptide. In one embodiment the method comprises: (a) culturing arecombinant host cell under conditions such that the polypeptide isexpressed; and (b) recovering the polypeptide.

Yet a further embodiment is an isolated rat ACC2 polypeptide. Forexample, the polypeptide comprises an amino acid sequence selected fromthe group consisting of: (a) a polypeptide comprising SEQ ID NO:14; (b)a polypeptide comprising amino acids 2 to 2455 of SEQ ID NO:14, whereinamino acids 2 to 2455 comprise a polypeptide of SEQ ID NO:14 minus thestart methionine; and (c) a polypeptide comprising amino acids 1 to 2455of SEQ ID NO:14. In another embodiment, the polypeptide comprises two ormore sequential amino acid deletions from one or both of: (a) theCOOH-terminus of the polypeptide; and (b) the NH₂-terminus of thepolypeptide.

Further disclosed is a method of identifying a compound that modulatesthe activity of a rat ACC2 polypeptide. In one embodiment, the methodcomprises: (a) determining the activity of a polypeptide in the absenceof a test compound; (b) determining the activity of the polypeptide inthe presence of a test compound; and (c) comparing the activity of thepolypeptide in the presence of the test compound with the activity ofthe polypeptide in the absence of the test compound, wherein a change inthe activity of the polypeptide in the presence of the test compoundrelative to the activity of the polypeptide in the absence of the testcompound indicates that the compound that modulates the activity of thepolypeptide.

An isolated antibody that specifically binds to the rat ACC2 polypeptideis disclosed. In various embodiments, the antibody is selected from thegroup consisting of a chimeric antibody, a single chain antibody, a Fabfragment, and a humanized antibody.

A method of isolating a rat ACC polypeptide is additionally disclosed.In one embodiment, the method comprises: (a) contacting crude lysatederived from a cell or tissue expressing an ACC polypeptide with anantibody to form a complex comprising an antibody and an ACC; (b)washing the complex with a buffer comprising 0.5 M NaCl; and (c)contacting the complex with an eluting ligand. The antibody can comprisean IgG antibody, and in one embodiment, can be, for example, a c-Myc-5IgG antibody and the eluting ligand can be a myc peptide. The mycpeptide can comprise the amino acid sequence of SEQ ID NO:16. In anotherexample, the IgG antibody is an anti-FLAG IgG antibody. Further, theantibody can be bound to a substrate. The method can be employed toisolate any ACC polypeptide, such as an ACC1 or ACC2 polypeptide.

Additionally, a polynucleotide capable of inhibiting the expression ofan ACC2 gene comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs:11 and 13 by antisense inhibition isdisclosed, as well as a method of inhibiting ACC2 gene expressioncomprising introducing an antisense polynucleotide into a cell or tissuethat expresses an ACC2 gene, thereby inhibiting the expression of thegene in the cell or tissue by antisense inhibition.

A polynucleotide capable of inhibiting the expression of an ACC2 genecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NOs:11 and 13 by RNA inhibition is disclosed, as well as amethod of inhibiting ACC2 gene expression comprising introducing an RNAipolynucleotide into a cell or tissue that expresses an ACC2 gene,thereby inhibiting the expression of the gene in the cell or tissue byRNA inhibition.

Another embodiment is directed to an isolated polypeptide comprising anACC2 polypeptide encoded by the cDNA deposited as ATCC Accession No.PTA-6054.

An additional embodiment is a method of increasing the activity of ahuman ACC2 polypeptide. In one embodiment, the method comprisesgenerating an enhanced ACC2 polypeptide comprising: (a) a phenylalanineresidue at position 254, (b) a glutamine residue at position 346, (c) athreonine residue at position 565, (d) an asparagine at position 841,(e) a valine residue at position 1103, (f) a cysteine residue atposition 1259, (g) an alanine residue at position 1526, and (h) anisoleucine residue at position 1717, wherein the human ACC2 polypeptidedoes not comprise SEQ ID NO:12 and wherein the enhanced ACC2 polypeptidehas an enzymatic activity level that is greater than the enzymaticactivity level of an ACC2 polypeptide that does not contain theindicated residues at the indicated positions. In various aspects, thehuman ACC2 polypeptide sequence is selected from the group consisting ofSEQ ID NOs:2, 4 and 6.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1K depicts a polynucleotide (SEQ ID NO:11) and encoded ACC2amino acid sequence (SEQ ID NO:12) of a human ACC2.

FIGS. 2A-2K depicts a polynucleotide (SEQ ID NO:13) and encoded ACC2amino acid sequence (SEQ ID NO:14) of a rat ACC2 identified as describedherein.

FIGS. 3A-3I is an alignment of a published rat nucleotide ACC2 sequence(SEQ ID NO:9) with a rat ACC2-encoding sequence consensus sequencedescribed herein (SEQ ID NO:13). In the figure, “AB004329” representsthe nucleic aside sequence of GenBank Accession No. AB004329 and “BMS”represents a rat ACC2 sequence.

FIGS. 4A-4C is an alignment of a published rat amino acid ACC2 sequence(SEQ ID NO:10) with a rat ACC2-encoding sequence consensus sequencedescribed herein (SEQ ID NO:14). In the figure, “AB004329” representsthe amino acid sequence of GenBank Accession No. AB004329 and“BMS_ratACC2” represents the amino acid sequence of a rat ACC2 sequencedescribed herein.

FIGS. 5A-5I is an alignment of a cloned rat ACC2 nucleotide sequence(SEQ ID NO:13) with a sequence derived from PCR products generated inconsensus sequencing (SEQ ID NO:15). In the figure, “ratACC2_C7”represents the cloned rat ACC2 nucleotide sequence and “RatACC2”represents the nucleotide sequence derived from PCR products generatedin consensus sequencing.

FIG. 6A is a schematic drawing for comparison of the primary structureof human ACC1 versus ACC2 and the final version construct of human ACC2which was used for expression in the work described herein; BCrepresents the biotin carboxylase domain, BCCP represents the biotincarboxyl carrier protein domain, CT represents the carboxyltransferasedomain, filled circles denote the biotin group, open circles denote thediscrepancies of amino acids between pYES-human-ACC2 (designated as Mt)versus the wild type human ACC2 (designated as WT), V5, Myc, 6Hisrepresent three tags fused in frame to the human ACC2 sequence at theCOOH-terminus and the numbers presented below the bar denote the aminoacid numbers predicted by the full length human ACC2 cDNA.

FIG. 6B is an autoradiograph depicting the results of blot analyses oftotal cell extracts from Sf9 cells that are infected with either wildtype baculovirus as Mock control, or with ACC2Mt and ACC2WT recombinantvirus respectively; the blots were probed with anti-V5 IgG (left panel)or with Streptavidin-HRP-conjugated (right panel) respectively.

FIG. 7 is an autoradiograph depicting the results of a chromatographicseparation of recombinant human ACC2 on monomeric avidin column.

FIG. 8 is a photograph (left panel) and an autoradiograph (right panel)depicting the results of a chromatographic separation of total Sf9 celllysates and recombinant human ACC2 on TALON resin assayed by coomassiestain (left panel) or by anti-V5 immunoblot analysis (right panel).

FIG. 9A is a photograph depicting the results of a chromatographicseparation of a recombinant human ACC2 of the present invention on ac-Myc-5 affinity column; fractions eluted from the column by myc peptide(SEQ ID NO:16) were assayed with coomassie stain.

FIG. 9B is a bar graph depicting the results of a chromatographicseparation of a recombinant human ACC2 of the present invention on ac-Myc-5 affinity column; fractions eluted from the column by myc peptide(SEQ ID NO:16) were assayed with ACC activity measurement.

FIG. 10 is a series of four plots depicting the concentration dependenceof a recombinant human ACC2 described herein, on the substrates acetylCoA, bicarbonate, ATP and its effector citrate; each plot is labeledaccording to substrate.

FIG. 11 is a series of two plots depicting the concentration dependentinhibition of a recombinant human ACC2 described herein by the knowninhibitors of ACC enzymes palmitoyl CoA and malonyl CoA; each plot islabeled according to inhibitor.

DETAILED DESCRIPTION OF THE INVENTION

The subject matter disclosed and claimed herein relates to novelnucleotide sequences encoding human and rat ACC2 proteins and to thenovel proteins themselves. Several embodiments are described and includeuses of the novel sequences for identifying modulators of ACC and fortreating conditions associated with undesired ACC activity. The novelsequences disclosed herein are consensus sequences that were identified,cloned and sequenced based on published versions of the human and ratACC2 sequences.

A human ACC2 polynucleotide sequence of the present invention is setforth in FIG. 1 (SEQ ID NO:11), and a rat ACC2 polynucleotide of thepresent invention is set forth in FIG. 2 (SEQ ID NO:13). The humansequence was deposited with ATCC on Jun. 8, 2004 and has been assignedDeposit Number PTA-6054. A human ACC2 polypeptide sequence of thepresent invention is set forth in FIG. 1 (SEQ ID NO:12), and a rat ACC2polypeptide of the present invention is set forth in FIG. 2 (SEQ IDNO:14). Based on the established physiological function of ACC2, thesenovel sequences represent an important target for the treatment ofobesity, diabetes and related disease states.

I. Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of ±20% or less (e.g., ±15%, ±10%, ±7%,±5%, ±4%, ±3%, ±2%, ±1%, or ±0.1%) from the specified amount, as suchvariations are appropriate.

As used herein, unless clearly specified otherwise explicitly or bycontext, the terms “ACC2” and “ACC2 of the present invention” are usedinterchangeably and mean an acetyl CoA carboxylase polypeptidecomprising SEQ ID NOs:12 or 14, which can be encoded by a polynucleotidesequence comprising the nucleic acid sequence of SEQ ID NO:11 (humanACC2) or SEQ ID NO:13 (rat ACC2). The terms also encompass variants,such as, but not limited to, polynucleotides that are not identical toSEQ ID NOs:11 and 13, due to degeneracy in the genetic code, but stillcode for an ACC2 polypeptide.

The terms “ACC2” and “ACC2 of the present invention” whether referringto a rat or a human sequence, encompasses sequences comprising one ormore conservative substitutions in the ACC2 amino acid sequences of SEQID NOs:12 and 14. The substitution can be naturally occurring orintroduced by man. In a conservative substitution, the replacement groupwill have approximately the same size, shape, hydrophobicity and chargeas the original group. A table disclosing some representative, butnon-limiting properties that can be used as a guide when identifying orgenerating a conservative mutation follows: Representative ConservativeAmino Acid Substitutions Amino Acid Property Amino Acid Basic: argininelysine histidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Conservative substitutions typically include the substitution of oneamino acid for another with similar characteristics, e.g., substitutionswithin the following groups: valine, glycine; glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. Other conservative amino acid substitutions are shown in thefollowing table: For Amino Acid Code Replace with any of: Alanine AD-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine ND-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic D D-Asp, D-Asn, Asn,Glu, D-Glu, Acid Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr,D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic ED-Glu, D-Asp, Asp, Asn, D-Asn, Acid Gln, D-Gln Glycine G Ala, D-Ala,Pro, D-Pro, .beta.- Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu,Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg,D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-OrnMethionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylal- F D-Phe, Tyr, D-Thr, L-Dopa, His, anine D-His, Trp, D-Trp,Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline PD-Pro, L-1-thioazolidine-4- carboxylic acid, D- or L-1-oxazo-lidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met,D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser,allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr,Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile,Met, D-Met

As used herein, the term “agonist,” and grammatical derivations thereof,refer to an agent that initiates, supplements or potentiates thebioactivity of a functional ACC2 gene or protein, or that supplements orpotentiates the bioactivity of a naturally occurring or engineeredfunctional ACC2 gene or protein. An agonist can be a ligand. Further, anagonist can act by preventing an antagonist from acting on a givenprotein.

As used herein, the terms “amino acid,” “amino acid residue” and“residue” are used interchangeably and mean any of the twenty naturallyoccurring amino acids. An amino acid is formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide (e.g., enzymatic activity). NH₂ refers tothe free amino group present at the amino terminus of a polypeptide.COOH refers to the free carboxy group present at the carboxy terminus ofa polypeptide. In addition, the phrases “amino acid” and “amino acidresidue” are broadly defined to include modified and unusual aminoacids.

As used herein, the term “antagonist,” and grammatical derivationsthereof, means an agent that decreases or inhibits the bioactivity of afunctional ACC2 gene or protein, or that decreases or inhibits thebioactivity of a naturally occurring or engineered ACC2 gene or protein.An antagonist can be a ligand. Further, an antagonist can act bypreventing an agonist from acting on a given protein.

As used herein, the term “antibody” means polyclonal, monoclonal,antibody fragments (e.g. a Fab fragment) and antibody derivatives. Theterm encompasses antibodies prepared by recombinant techniques, such aschimeric or humanized antibodies, as well as single chain or bispecificantibodies. The term specifically encompasses antibodies that bind to anepitope, or a portion thereof, of a polypeptide that is described in thepresent disclosure.

As used herein, the terms “antigen” and “epitope,” which are wellunderstood in the art, mean all or a portion of a macromolecule that isspecifically recognized by a component of the immune system, e.g. anantibody or a T-cell antigen receptor. An epitope is a region of anantigen. As used herein, the term “antigen” encompasses antigenicepitopes, e.g., fragments of an antigen that are antigenic epitopes.

As used herein, the term “associates specifically,” and grammaticalderivations thereof, means an interaction between a first moiety (e.g. amodulator, such as an agonist or an antagonist) and a second moiety(e.g., an ACC2 polypeptide or fragment thereof) that occurspreferentially to an interaction the first or second moiety and anyother moieties present. For example, an antibody is presented with avariety of antigens, but only binds to a particular antigen. In thisexample, the antibody “specifically associates” with the particularantigen.

As used herein, the term “biological activity” means any activity that abiological molecule normally exhibits in vivo. For example, when thebiological molecule is ACC2, representative biological activities caninclude the catalytic carboxylation of biotin covalently bound to ACC2polypeptide in an ATP-dependent manner, the catalytic formation ofmalonyl CoA as a result of transfer of carboxyl group to acetyl CoA, andthe binding of citrate.

As used herein, the term “biological sample” means any biological sampleobtained from an organism, body fluids, cell line, tissue culture. Abiological sample can be a body fluid (for example, sputum, amnioticfluid, urine, saliva, breast milk, secretions, interstitial fluid,blood, serum, spinal fluid, etc.) or other tissue source. Methods forobtaining tissue biopsies and body fluids from organisms are known tothose of ordinary skill in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is a preferred source.

As used herein the term “complementary” means a nucleic acid sequencethat is base paired, or is capable of base-pairing, according to thestandard Watson-Crick complementarity rules. These rules generally holdthat guanine pairs with cytosine (G:C) and adenine pairs with eitherthymine (A:T) in the case of DNA, or adenine pairs with uracil (A:U) inthe case of RNA.

As used herein, the term “hybridize” means the process by which apolynucleotide strand anneals with a complementary strand through basepairing under defined hybridization conditions. Specific hybridizationis an indication that two nucleic acid sequences share a high degree ofcomplementarity.

As used herein, the terms “isolated” and “purified” are usedinterchangeably and refer to material (e.g., a nucleic acid or apolypeptide) removed from its original environment (e.g., the naturalenvironment, if it is naturally occurring), and thus is altered “by thehand of man” from its natural state. The term “isolated” does not referto genomic or cDNA libraries, whole cell total or mRNA preparations,genomic DNA preparations (including those separated by electrophoresisand transferred onto blots), sheared whole cell genomic DNA preparationsor other compositions where the art demonstrates no distinguishingfeatures of the polynucleotide and/or protein sequences of the presentinvention; such sequences are excluded from the scope of the presentinvention.

As used herein the term “modulate,” and grammatical derivations thereof,refer to an increase, decrease, or other alteration of any and/or allchemical and/or biological activities or properties mediated by a givenDNA sequence, RNA sequence, polypeptide, peptide or molecule. Thedefinition of “modulator” as used herein encompasses agonists and/orantagonists of a particular activity or protein. The term “modulate”refers to both upregulation (i.e., activation or stimulation) anddownregulation (i.e. inhibition or suppression).

As used herein, the term “stringent hybridization conditions,” in thecontext of nucleic acid hybridization experiments such as southern andnorthern blot analysis, means a set of conditions under which singlestranded nucleic acid sequences are unlikely to hybridize to one anotherunless there is substantial complementarity between the sequences.Stringent hybridization conditions can be both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found, for example, in Tijssen, (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, Elsevier, N.Y. Generally, highly stringenthybridization and wash conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. Typically, under “stringent conditions” aprobe will hybridize specifically to its target subsequence, but to noother sequences.

Examples of stringency conditions are shown in the table below: highlystringent conditions are those that are at least as stringent as, forexample, conditions A-F; stringent conditions are at least as stringentas, for example, conditions G-L; and reduced stringency conditions areat least as stringent as, for example, conditions M-R. StringencyConditions Hybridization Wash Stringency Polynucleotide Hybrid LengthTemperature Temperature Condition Hybrid± (bp) ‡ and Buffer† and Buffer†A DNA:DNA > or equal to 50 65° C.; 1xSSC - 65° C.; 0.3xSSC or- 42° C.;1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > orequal to 50 67° C.; 1xSSC - 67° C.; 0.3xSSC or- 45° C.; 1xSSC, 50%formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 5070° C.; 1xSSC - 70° C.; 0.3xSSC or- 50° C.; 1xSSC, 50% formamide FRNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 50 65° C.;4xSSC - 65° C.; 1xSSC or- 45° C.; 4xSSC, 50% formamide H DNA:DNA <50Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 50 67° C.; 4xSSC - 67° C.;1xSSC or- 45° C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*;4xSSC K RNA:RNA > or equal to 50 70° C.; 4xSSC - 67° C.; 1xSSC or- 40°C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA >or equal to 50 50° C.; 4xSSC - 50° C.; 2xSSC or- 40° C. 6xSSC, 50%formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to 5055° C.; 4xSSC - 55° C.; 2xSSC or- 42° C.; 6xSSC, 50% formamide P DNA:RNA<50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 50 60° C.; 4xSSC - 60°C.; 2xSSC or- 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*; 4xSSC Tr*;4xSSC‡ The “hybrid length” is the anticipated length for the hybridizedregion(s) of the hybridizing polynucleotides. When hybridizing apolynucleotide of unknown sequence, the hybrid is assumed to be that ofthe hybridizing polynucleotide of the present invention. Whenpolynucleotides of known sequence are hybridized, the hybrid length canbe determined by# aligning the sequences of the polynucleotides and identifying theregion or regions of optimal sequence complementarity. Methods ofaligning two or more polynucleotide sequences and/or determining thepercent identity between two polynucleotide sequences are well known inthe art (e.g., MEGALIGN program of the DNA*Star suite of programs, etc).†SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4)can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodiumcitrate) in the hybridization and wash buffers; washes are performed for15 minutes after hybridization is complete. The hybridizations andwashes may additionally include 5× Denhardt's reagent, 0.5-1.0% SDS, 100μg/ml# denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, andup to 50% formamide.*Tb − Tr: The hybridization temperature for hybrids anticipated to beless than 50 base pairs in length should be 5-10° C. less than themelting temperature Tm of the hybrids there Tm is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases).# For hybrids between 18 and 49 base pairs in length, Tm(° C.) = 81.5 +16.6(log₁₀[Na⁺]) + 0.41(% G + C) − (600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1xSSC = .165 M).

±—The present invention encompasses the substitution of any one, or moreDNA or RNA hybrid partners with either a PNA, or a modifiedpolynucleotide. Such modified polynucleotides are known in the art andare more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, ColdSpring Harbor (2001), and Current Protocols in Molecular Biology, 1995,F. M., Ausubel et al., eds, John Wiley and Sons, Inc., which are herebyincorporated by reference herein.

As used herein, the term “vector” means a replicon, such as plasmid,phage or cosmid, to which another DNA segment may be attached so as tobring about the replication of the attached segment.

II. ACC2 Polypeptides

ACC2 polypeptides that form aspects of the present invention arepresented in FIGS. 1 and 2 and in SEQ ID NOs:12 and 14. These sequencesrepresent human and rat genomic ACC2 polypeptide sequences. Althoughseveral human and rat ACC2 sequences have been published (see, e.g.,Human ACC2: GenBank Accession No. NM_(—)001093 (SEQ ID NOs:1 and 2),GenBank Accession No. AC007637 (SEQ ID NOs:3 and 4) and the sequence ofpYES-human ACC2, (SEQ ID NOs:5 and 6); Rat ACC2: GenBank Accession No.NM_(—)053922 (SEQ ID NOs:7 and 8) and GenBank Accession No. AB004329(SEQ ID NOs:9 and 10)), there are discrepancies between these sequences.Consequently, the present inventors re-evaluated the published human andrat ACC2 sequences, which lead to the identification of the ACC2sequences of the present invention.

Based on the identified discrepancies in the published sequences, it wasspeculated that these discrepancies may represent inadvertent mutationsintroduced during a cloning or sequencing process. The positions atwhich residues differ between the published human and rat sequences werealso identified. In the human ACC2 amino acid consensus sequence of thepresent invention, these positions are occupied by R at position 9, P atposition 111, A at position 127, F at position 254, Q at position 345, Vat position 347, AGWG at positions of 349-352, P at position 450, T atposition 565, H at position 614, E at position 656, E at position 671,ET at position 742-743, E at position 799, N at position 841, V atposition 1025, V at position 1064, V at position 1103, C at position1259, R at position 1480, A at position 1526, R at position 1547, I atposition 1717, G at position 1821, I at position 2141, PPYA at position2194-2197, and K at position 2242. In the rat ACC2 amino acid consensussequence of the present invention, these positions in the sequence areoccupied by residues C at position 9, K at position 30, S at position42, S at position 50, S at position 91, H at position 153, A at position178, S at position 179, A at position 191, L at position 196, C atposition 272, I at position 308, QYV at position 313-315, E at position365, PSEA at position 372-375, WA at position 377-378, KI at position382-383, P at position 422, R at position 463, ML at position 472-473, Tat position 534, G at position 556, E at position 563, G at position658, AD at position 693-694, R at position 707, F at position 742, C atposition 774, M at position 788, L at position 849, K at position 940, Lat position 1025, G at position 1048, M at position 1062, Y at position1065, Y at position 1122, P at position 1159, IFLSAIDMY at position1243-1251, R at position 1467, A at position 1493, PT at position1596-1597, E at position 1629, PK at position 1737-1738, RM at position1832-1833, RYV at position 1890-1892, T at position 1932, A at position2079, D at position 2111, P at position 2150, Y at position 2168, F atposition 2185, A at position 2203, GQL at position 2260-2262, TA atposition 2264-2265, E at position 2309, I at position 2403, and DCVA atposition 2428-2431. Details regarding the analysis of the published ACC2sequences and the generation of the human and rat ACC2 polypeptidesequences of the present invention are provided in the accompanyingExamples. Methods of isolating and using the polypeptides are alsoprovided.

Although the ACC2 polypeptide sequences of SEQ ID NOs:12 and 14 form anaspect of the present invention, the polypeptides of the presentinvention are not limited to the precise sequences provided in theSequence Listing. In other aspects of the present invention, variantpolypeptides, and polypeptides comprising non-standard amino acids, canbe generated using techniques known to those of ordinary skill in theart.

The polypeptides of the present invention can comprise non-standardamino acids, namely amino acids other than the 20 gene-encoded aminoacids. Such polypeptides can be generated by natural processes, such asby posttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are described inbasic texts and in more detailed monographs, as well as in the pertinentresearch literature. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini.

A given polypeptide can contain many types of modifications.Polypeptides can be branched, for example, as a result ofubiquitination, and they can be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides can result fromposttranslation natural processes or can be made by employing syntheticmethods known to those of ordinary skill in the art. Representativemodifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Tags to facilitatepurification can also be added (see, e.g. Creighton, Proteins—Structuresand Molecular Properties, 2nd ed., W.H. Freeman, New York (1992);Posttranslational Covalent Modification of Proteins, (Johnson, ed.),Academic Press, New York, (1984); Seifter & Englard, Method Enzymol.182:626-646 (1990)).

The ACC2 polypeptides of the present invention, and variants, fragmentsand serial deletions thereof, can be produced by any method known in theart for the synthesis of polypeptides, for example, by chemicalsynthesis, by the recombinant expression techniques described herein orby purification from a biological source, such as tissue, as describedherein. For example, methods that are well known to those skilled in theart can be used to construct expression vectors containing a partial orthe entire native or mutated ACC2 polypeptide coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, as described herein,synthetic techniques and in vivo recombination/genetic recombination.

III. Polynucleotides

ACC2 polynucleotides that form aspects of the present invention arepresented in FIGS. 1 and 2 and in SEQ ID NOs:11 and 13. In the case ofhuman ACC2, the present inventors have found discrepancies between thepublished sequences and the sequences of the present invention and havereconciled these differences in the ACC2 nucleotide and polypeptidesequences of the present invention.

The present inventors have identified discrepancies between thepublished ACC2 sequences. These residue may comprise inadvertentmutations introduced by the cloning process. In the human ACC2 sequenceof the present invention, these positions in the sequence are occupiedby R at position 9, P at position 111, A at position 127, F at position254, Q at position 345, V at position 347, AGWG at positions of 349-352,P at position 450, T at position 565, H at position 614, E at position656, E at position 671, ET at position 742-743, E at position 799, N atposition 841, V at position 1025, V at position 1064, V at position1103, C at position 1259, R at position 1480, A at position 1526, R atposition 1547, I at position 1717, G at position 1821, I at position2141, PPYA at position 2194-2197, and K at position 2242. In the ratACC2 sequence of the present invention, these positions in the sequenceare occupied by C at position 9, K at position 30, S at position 42, Sat position 50, S at position 91, H at position 153, A at position 178,S at position 179, A at position 191, L at position 196, C at position272, I at position 308, QYV at position 313-315, E at position 365, PSEAat position 372-375, WA at position 377-378, KI at position 382-383, Pat position 422, R at position 463, ML at position 472-473, T atposition 534, G at position 556, E at position 563, G at position 658,AD at position 693-694, R at position 707, F at position 742, C atposition 774, M at position 788, L at position 849, K at position 940, Lat position 1025, G at position 1048, M at position 1062, Y at position1065, Y at position 1122, P at position 1159, IFLSAIDMY at position1243-1251, R at position 1467, A at position 1493, PT at position1596-1597, E at position 1629, PK at position 1737-1738, RM at position1832-1833, RYV at position 1890-1892, T at position 1932, A at position2079, D at position 2111, P at position 2150, Y at position 2168, F atposition 2185, A at position 2203, GQL at position 2260-2262, TA atposition 2264-2265, E at position 2309, I at position 2403, and DCVA atposition 2428-2431. Details regarding the generation of the human andrat ACC2 sequences of the present invention are provided in theaccompanying Examples.

In one aspect of the present invention, isolated polynucleotidesencoding a polypeptide comprising the amino acid sequence of human ACC2(SEQ ID NO:12) and rat ACC2 (SEQ ID NO:14) is disclosed. Examples ofsuch polynucleotides are presented in FIGS. 1 and 2 and in SEQ ID NOs:11and 13, which encode human and rat ACC2 proteins, respectively. Inanother aspect of the present invention, the nucleotide sequence of thecDNA insert of the plasmid deposited with the ATCC as Accession NumberPTA-6054 on Jun. 8, 2004, and complements thereof, are disclosed.

The present invention encompasses complements of the ACC2-encodingpolynucleotides of the present invention. As explained herein, acomplementary sequence is a nucleotide sequence that it can hybridize toa polynucleotide sequence of the present invention to form a stableduplex. Sequences that are complementary to an ACC2 polynucleotidesequence of the present invention can be readily identified using thesequences provided in SEQ ID NOs:11 and 13 as templates. Thus, thepresent invention encompasses not only polynucleotide sequences encodingthe ACC2 proteins of the present invention, but complements of thesesequences as well.

As used herein, a “polynucleotide” of the present invention includes thepolynucleotides disclosed herein and in the Sequence Listing, as well asthose polynucleotides capable of hybridizing, under stringenthybridization conditions, to the polynucleotide sequences of SEQ IDNOs:11 and 13, the complement thereof, or the cDNA within the clonedeposited with the ATCC. “Stringent hybridization conditions” aredescribed herein.

A polynucleotide which hybridizes only to polyA+ sequences (such as any3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or toa complementary stretch of T (or U) residues, is not included in thedefinition of “polynucleotide,” since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., almost any double-stranded cDNA clonegenerated using oligo dT as a primer).

The polynucleotides of the present invention can comprise anypolyribonucleotide or polydeoxyribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides cancomprise single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can comprise triple-strandedregions comprising RNA, DNA or both RNA and DNA. A polynucleotide canalso contain one or more modified bases or DNA or RNA backbones modifiedfor stability or for other reasons. “Modified” bases include, forexample, tritylated bases and unusual bases such as inosine. A varietyof modifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

The polynucleotides of the present invention are useful as probes forthe identification and isolation of full-length cDNAs and/or genomic DNAwhich correspond to the polynucleotides of the present invention, asprobes to hybridize to and discover novel, related DNA sequences, asprobes for positional cloning of a sequence of the present invention ora related sequence, as probe to “subtract-out” known sequences in theprocess of discovering other novel polynucleotides, as probes toquantify polynucleotide expression, and as probes for microarrays.

The present invention further provides for other experimental methodsand procedures currently available to derive functional assignments.These procedures include but are not limited to spotting of clones onarrays, micro-array technology, PCR based methods (e.g., quantitativePCR), anti-sense methodology, gene knockout experiments, and otherprocedures that could use sequence information from clones to build aprimer or a hybrid partner.

Details regarding the generation of the human and rat ACC2 sequences ofthe present invention are provided herein and in the accompanyingExamples.

IV. Fragments

The present invention encompasses fragments of the polynucleotidesencoding ACC2 proteins of the present invention. As used herein, theterm “fragment” means a polynucleotide sequence that is shorter than anACC2-encoding polynucleotide sequence of the present invention (e.g.,SEQ ID NOs:11 and 13), but retains a region comprising the contiguoussequence of the polynucleotide from which the fragment is derived. Afragment of an ACC2 nucleotide sequence can encode a biologically activeportion of an ACC2 protein, or it can be a fragment that can be used asa hybridization probe or as a primer. Nucleic acid molecules that arefragments of an ACC2-encoding polynucleotide can comprise any number ofnucleotides up to the number of nucleotides present in a full-lengthACC2-encoding polynucleotide sequence of the present invention.

The term “fragment,” therefore, includes any contiguous sequence notdisclosed prior to the present invention, but excludes sequences knownprior to the present invention. More particularly, if an isolatedfragment is disclosed prior to the present invention, that fragment isnot encompassed by the present invention.

A fragment of an ACC2-encoding polynucleotide sequence can, but neednot, encode a biologically active ACC2. For example a fragment of anACC2-encoding polynucleotide sequence of the present invention can beemployed as a probe or as a primer, in which case, these fragments willnot encode a biologically active protein. On the other hand, a truncatedform of an ACC2-encoding polynucleotide sequence of the presentinvention may encode a biologically active protein, yet be referred toas a fragment. Both biologically active and non-biologically activesequences are within the scope of the claims of the present invention.

Polypeptide fragments also form aspects of the present invention. Apolypeptide fragment of the present invention can comprise any number ofamino acids up to the full length of an ACC2 amino acid sequence of thepresent invention. Polypeptide fragments of the present inventioninclude truncations of any length up to, but excluding, a full lengthACC2 polypeptide of the present invention. As discussed herein, apolypeptide fragment of the present invention excludes sequences knownprior to the present invention. Thus, an isolated fragment that wasdescribed prior to the present invention, is not encompassed by thepresent invention.

A polypeptide fragment can be, for example, an epitope, which can beemployed to raise antibodies against an ACC2 polypeptide of the presentinvention, as described herein and as generally known to those ofordinary skill in the art. Fragments can, but need not, include abiologically active segment of an ACC2 protein of the present invention.Other applications for the polypeptide fragments of the presentinvention will be apparent to those of ordinary skill in the art.

A polypeptide fragment of the present invention can comprise an ACC2sequence of the present invention from which serial deletions from theNH₂- or COOH-terminus, or both the NH₂- and COOH-terminus have beenmade. Such a fragment will comprise a variable number of contiguousamino acids of an ACC2 polypeptide of the present invention, but will beshorter in sequence than a full-length ACC2 polypeptide of the presentinvention.

V. Variants

In a further aspect of the present invention, the polypeptides andpolynucleotides of the present invention encompass sequences that arevariants of the ACC2 polypeptide and ACC2-encoding polynucleotidesequences disclosed herein. As used herein, a “variant polynucleotide”is a polynucleotide or polypeptide differing from the polynucleotide orpolypeptide of the present invention, but retaining essential propertiesthereof. Generally, variants are similar, and, over many regions,identical to the polynucleotide or polypeptide of the present invention.“Variants” of the polynucleotide sequences of the present inventioninclude sequences that encode an ACC2 protein of the present invention,but differ conservatively because of the degeneracy of the genetic code.These naturally occurring variants can be identified using standardmethodology, such as polymerase chain reaction (PCR), hybridization andsequencing techniques.

A variant can comprise alterations in the coding regions, non-codingregions, or both regions of an ACC2-encoding polynucleotide sequence Forexample, a variant can comprise alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. As noted, nucleotide variantsproduced by silent substitutions due to the degeneracy of the geneticcode are within the scope of the claims. Moreover, a variant cancomprise any number of amino acid substitutions, for example 1, 2, 3, 4,5, 7, 8, 10, or more amino acids can be substituted, deleted, or addedin any combination are also within the scope of the claims. Preferably,a variant of the present invention retains the amino acids identified asdifferent from published ACC2 sequences (i.e., R at position 9, P atposition 111, A at position 127, F at position 254, Q at position 345, Vat position 347, AGWG at positions of 349-352, P at position 450, T atposition 565, H at position 614, E at position 656, E at position 671,ET at position 742-743, E at position 799, N at position 841, V atposition 1025, V at position 1064, V at position 1103, C at position1259, R at position 1480, A at position 1526, R at position 1547, I atposition 1717, G at position 1821, I at position 2141, PPYA at position2194-2197, and K at position 2242 in the human sequence and C atposition 9, K at position 30, S at position 42, S at position 50, S atposition 91, H at position 153, A at position 178, S at position 179, Aat position 191, L at position 196, C at position 272, I at position308, QYV at position 313-315, E at position 365, PSEA at position372-375, WA at position 377-378, KI at position 382-383, P at position422, R at position 463, ML at position 472-473, T at position 534, G atposition 556, E at position 563, G at position 658, AD at position693-694, R at position 707, F at position 742, C at position 774, M atposition 788, L at position 849, K at position 940, L at position 1025,G at position 1048, M at position 1062, Y at position 1065, Y atposition 1122, P at position 1159, IFLSAIDMY at position 1243-1251, Ratposition 1467, A at position 1493, PT at position 1596-1597, E atposition 1629, PK at position 1737-1738, RM at position 1832-1833, RYVat position 1890-1892, T at position 1932, A at position 2079, D atposition 2111, P at position 2150, Y at position 2168, F at position2185, A at position 2203, GQL at position 2260-2262, TA at position2264-2265, E at position 2309, I at position 2403, and DCVA at position2428-2431).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are within the scope of thepresent invention. Alternatively, non-naturally occurring variants maybe produced by mutagenesis techniques or by direct synthesis. Nucleicacid molecules corresponding to natural allelic variants and homologuesof the ACC2 cDNA of the present invention can be isolated based on theiridentity to the polynucleotides disclosed herein using the cDNA, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions asdescribed herein.

Using known methods of protein engineering and recombinant DNAtechnology, variants can be generated to alter a characteristic of thepolypeptides of the present invention, such as molecular weight orantigenic response. For example, a variant can have one or more alteredcharacteristics while retaining other characteristics. For example, oneor more amino acids can be deleted from the NH₂-terminus orCOOH-terminus of the protein (as described herein), which will alter themolecular weight of the protein, and might alter the immunologicresponse profile of the variant and/or the preferred purificationprotocol for the variant, but might not substantially alter theenzymatic activity of the variant.

Even if deleting one or more amino acids from the NH₂-terminus orCOOH-terminus of a polypeptide results in modification or loss of one ormore biological functions, other biological activities might still beretained. For example, the ability of a deletion variant to induceand/or to bind antibodies which recognize the protein will likely beretained when less than the majority of the residues of the protein areremoved from the NH₂-terminus or COOH-terminus. Whether a particularpolypeptide lacking NH₂- or COOH-terminal residues of a protein retainssuch immunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Thus, the present invention encompasses polypeptide variants that showvarying degrees of biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

Various methods for making phenotypically silent amino acidsubstitutions are known. For example, by comparing amino acid sequencesin different species, conserved amino acids can be identified. Theseconserved amino acids are likely important for protein function. Incontrast, the amino acid positions where substitutions have beentolerated by natural selection indicates that these positions are notcritical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

In another example, standard mutagenesis methods can be employed tointroduce amino acid changes at specific positions of a cloned gene toidentify regions critical for protein function. For example, sitedirected mutagenesis or alanine-scanning mutagenesis (introduction ofsingle alanine mutations at every residue in the molecule) can beemployed. The resulting mutant molecules can then be tested forbiological activity.

Thus, the present invention encompasses, but is not limited to,conservative amino acid substitutions introduced into the ACC2polypeptides of the present invention. Particular examples ofconservative amino acid substitutions are presented herein.

In addition to conservative amino acid substitution, variants of thepresent invention include, but are not limited to: (i) substitutionswith one or more of the non-conserved amino acid residues, where thesubstituted amino acid residues may or may not be one encoded by thegenetic code, or (ii) substitution with one or more amino acid residueshaving a substituent group, (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as, forexample, an IgG Fc fusion region peptide, a leader or secretorysequence, or a sequence facilitating purification.

Methods of introducing coding and non-coding mutations into a sequenceare known in the art and can be readily employed in the presentinvention to generate polypeptide and polynucleotide variants (see, e.g.Sambrook et al. and Creighton).

VI. Recombinant Expression of ACC2 Sequences

In one aspect of the present invention, the ACC2 enzymes of the presentinvention can be expressed recombinantly. Thus, in accordance with thepresent invention, conventional molecular biology, microbiology,recombinant DNA and protein chemistry techniques known to those ofordinary skill of the art can be employed to produce a DNA sequenceencoding an ACC2 polypeptide, in addition to the guidance providedherein. Such techniques are explained fully in the relevant literature(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor (2001); Glover,DNA Cloning: A Practical Approach, (2^(nd) ed.) IRL Press, New York, USA(1995); Gait, Oligonucleotide Synthesis: A Practical Approach, IRLPress, New York, USA (1984); Hames & Higgins, Protein Expression: APractical Approach, Oxford University Press, New York, USA, (1999);Bickerstaff, Immobilization of Cells And Enzymes, Humana Press, Totowa,N.J., USA (1997); Perbal, A Practical Guide To Molecular Cloning (2^(nd)ed.) Wiley, New York, N.Y., USA (1988); Current Protocols in MolecularBiology, (Ausubel et al., eds.), Greene Publishing Associates andWiley-Interscience, New York (2002); Ausubel, Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, (4^(th) ed.) John Wiley & Sons, New York, N.Y., USA(1999)). A DNA sequence encoding an ACC2 polypeptide of the presentinvention (including variants, analogs, and functional equivalents), canbe prepared by various molecular biology methods known in the art.

Recombinant expression of an ACC2 polypeptide of the present invention,or a fragment, variant or analog thereof, (e.g. a human or a rat ACC2polypeptide), requires the construction of an expression vectorcomprising a polynucleotide that encodes the protein of interest. Once apolynucleotide encoding an ACC2 protein of the present invention hasbeen obtained, a vector for the production of the ACC2 polypeptide canbe produced by recombinant DNA technology using techniques known in theart. Methods for preparing a protein by expressing a polynucleotidecontaining an ACC2-encoding nucleotide sequence are known in the art anddescribed herein.

Methods known to those of ordinary skill in the art can be used toconstruct an expression vector comprising an ACC2 coding sequence andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The presentinvention, thus, encompasses replicable vectors comprising anACC2-encoding nucleotide sequence of the present invention, which may beoperably linked to a promoter.

The expression vector can then be transferred to a host cell byconventional techniques and the transfected cells are then culturedunder appropriate conditions to produce an ACC2 polypeptide of thepresent invention. Thus, the present invention also comprises host cellscontaining a polynucleotide encoding an ACC2 polypeptide of the presentinvention operably linked to a heterologous promoter.

A variety of host-expression vector systems can be employed to expressan ACC2 polypeptide of the present invention. Such host-expressionsystems represent vehicles by which a coding sequence of interest can beproduced and subsequently purified, but also represent cells that may,when transformed or transfected with the appropriate nucleotide codingsequences, express an ACC2 polypeptide of the present invention in situ.These include but are not limited to microorganisms such as bacteria(e.g. E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing ACC2 codingsequences; yeast (e.g. Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing ACC2 coding sequences;insect cell systems infected with recombinant virus expression vectors(e.g., baculovirus) containing ACC2 coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, (CaMV); tobacco mosaic virus, (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingACC2 coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK,293, 3T3 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Under someconditions it might be desirable that bacterial cells such asEscherichia coli, or eukaryotic cells be used for the expression of arecombinant ACC2 polypeptide.

In bacterial systems, a number of expression vectors can beadvantageously employed, depending upon the use intended for the ACC2polypeptide being expressed. For example, when a large quantity of sucha protein is to be produced, for example for the generation of apharmaceutical composition comprising an ACC2 polypeptide, vectors thatdirect the expression of high levels of fusion protein products that arereadily purified can be desirable. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orFactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) can be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The ACC2 polypeptide codingsequence may be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the ACC2 polypeptide coding sequence of interest can be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric gene canthen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing the ACC2 protein in infected hosts(see, e.g. Logan & Shenk, (1984) Proc. Natl. Acad. Sci. U.S.A.81:355-359). Specific initiation signals may also be required forefficient translation of inserted ACC2 coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. Furthermore,the initiation codon must be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe from a variety of origins, both natural and synthetic. The efficiencyof expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (see,e.g., Bittner et al., (1987) Method Enzymol. 153:51-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be employed.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theACC2 polypeptide can be engineered. Rather than using expression vectorsthat contain viral origins of replication, host cells can be transformedwith DNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express an ACC2 polypeptide. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that interact directly or indirectly with theACC2 polypeptide.

A number of selection systems can be employed in the recombinantexpression of an ACC2 polypeptide of the present invention. For example,the herpes simplex virus thymidine kinase (Wigler et al., (1977) Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, (1992) Proc. Natl. Acad. Sci. U.S.A. 48:202), and adeninephosphoribosyltransferase (Lowy et al., (1980) Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Additionally,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., (1980) Proc. Natl. Acad. Sci. U.S.A. 77:357; O'Hare et al.,(1981) Proc. Natl. Acad. Sci. U.S.A. 78:1527); gpt, which confersresistance to mycophenolic acid (Mulligan & Berg, (1981) Proc. Natl.Acad. Sci. U.S.A. 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu & Wu, (1991)Biotherapy 3:87-95; Tolstoshev, (1993) Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan, (1993) Science 260:926-932; and Morgan & Anderson,(1993) Ann. Rev. Biochem. 62:191-217; TIB TECH 11(5):155-215, May,1993); and hygro, which confers resistance to hygromycin (Santerre etal., (1984) Gene 30:147), to provide just a few examples.

Methods known in the art of recombinant DNA technology can be applied toselect the desired recombinant clone, and such methods are described,for example, in Current Protocols in Molecular Biology, (Ausubel et al.,eds.), Greene Publishing Associates and Wiley-Interscience, New York(2002); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York, N.Y., USA (1990); Current Protocols in HumanGenetics, (Dracopoli et al., eds.), John Wiley & Sons, New York, N.Y.,USA (1994), Chapters 12 and 13; and Colberre-Garapin et al., (1981) J.Mol. Biol. 150:1.

The expression levels of an ACC2 polypeptide can be increased in severaldifferent ways, for example by vector amplification (for a review ofthis technique, see e.g. Bebbington & Hentschel, in DNA Cloning, vol. 3,Academic Press, New York (1987)). When a marker in the vector systemexpressing an ACC2 polypeptide is amplifiable, an increase in the levelof inhibitor present in culture of host cell will increase the number ofcopies of the marker gene. Since the amplified region is associated withthe ACC2 gene, production of ACC2 protein will also increase.

Once an ACC2 polypeptide of the present invention has been produced by acell, tissue, animal, or has been chemically synthesized, orrecombinantly expressed, it can be purified by any method known in theart for purification of a polypeptide, for example, by chromatography(e.g., ion exchange, affinity, particularly by affinity for the specificantigen after Protein A, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. In addition, the ACC2polypeptides of the present invention, or fragments thereof, can befused to heterologous polypeptide sequences described herein orotherwise known in the art, to facilitate purification (e.g., a Histag). Another aspect of the present invention relates to thepurification of ACC polypeptides, which is not limited to ACC2polypeptides, is described herein and can be employed to generate anisolated ACC2 polypeptide.

VII. Isolation of ACC Polypeptides

The ACC2 polypeptides of the present invention can be isolated from anysuitable source, for example from cells recombinantly or endogenouslyexpressing the protein or from tissues such as rat livers and/or ratheart muscle. ACC2 can be isolated from a biological sample usingstandard protein purification methodology known to those of the art(see, e.g., Janson, Protein Purification: Principles, High ResolutionMethods, and Applications, (2^(nd) ed.) Wiley, New York, (1997);Rosenberg, Protein Analysis and Purification: Benchtop Techniques,Birkhauser, Boston, (1996); Walker, The Protein Protocols Handbook,Humana Press, Totowa, N.J., (1996); Doonan, Protein PurificationProtocols, Humana Press, Totowa, N.J., (1996); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York,(1994); Harris, Protein Purification Methods: A Practical Approach, IRLPress, New York, (1989)). Guidance in the isolation of an ACC and/or FASis provided herein, for example in the Examples (see, e.g. Example 9).Other methods of purifying active ACC will be known to those of skill inthe art and any such methods can be employed in the present invention.

In one aspect of the present invention, a method of isolating an ACCpolypeptide is provided. This purification method is exemplified in thecontext of ACC2, but the method can be employed to isolate any ACC(e.g., ACC1 or ACC2) polypeptide. In one embodiment, the methodcomprises a single step IgG mediated affinity column. The IgG mediatedaffinity column can comprise an anti-myc IgG. Anti-Myc-IgG, especiallyc-Myc-5 IgG, has been characterized (Hillman et al., (2001) ProteinExpression and Purification 23, 359-368) and can be readily prepared asdescribed by Hillman et al. (Hillman et al. (2001) Protein Expressionand Purification 23, 359-368).

In one embodiment of the method, total crude homogenized tissue or cellcytosolic lysates, for example lysates derived from ACC virus infectedSf9 cells or any other transfected cell, are loaded on a c-Myc-5 IgGcolumn. The loading can be accomplished by overnight incubation,although the precise loading procedure employed can depend on a varietyof factors, such as volume of lysate and whether any other pre-columnpurification procedures were performed (e.g., a preliminaryprecipitation, etc.). Such considerations will be known to those ofordinary skill in the art upon considering the present disclosure.

The bound protein can then extensive washed with high salt buffer. Abuffer comprising 0.5 M NaCl, for example can be employed. One purposeof this high salt wash is to remove a fraction of the proteins presentin the crude lysate; accordingly, the concentration and composition ofsalt employed in the wash can vary.

Following the salt wash, the proteins that remain bound to the columncan be eluted with an elution ligand. For example, when an anti-myc IgGantibody is employed, the elution ligand can be a peptide comprising amyc peptide (EQKLISEEDL; SEQ ID NO:16), which can comprise variousmodifications, such capping of the NH₂- or COOH-terminal, whicheliminate charges. The elution of the ACC protein from the column can beperformed stepwise, for example in a series of steps (e.g., 1, 2, 5, 7,10 or more steps). Additional suitable elution ligands can be employedand can depend, in part, on the nature of the IgG employed in themethod. In another example, when an anti-FLAG IgG is employed theelution ligand can comprise a FLAG peptide, which comprises the coresequence DYKD (SEQ ID NO:33) and is commercially available as a peptidehaving the sequence DYKDDDDK (SEQ ID NO:34).

The presence of ACC in the eluent of each step can be confirmed byimmunological techniques and/or by simply running the eluent on an SDSPAGE gel and identifying the molecular weight of the eluted protein.Additionally, the identity of the eluted protein can be confirmed by anenzymatic assay, such as that described herein (see also U.S. PatentApplication Ser. No. 60/558,015, incorporated herein by reference in itsentirety) or an assay known in the art (see, e.g., Waite & Wakil (1962)J. Biol. Chem. 237:2750-2757, Tanabe et al., (1981) Methods Enzymol. 71Pt C, 5-16; Wakil et al., (1959) Biochim. Biophys. Acta 34:227-233, alsoincorporated herein by reference). The eluent from each step can beevaluated separately and the active fractions can subsequently bepooled. Pooled protein can be stored in buffer or in a lyophilized form.

VIII. Methods of Assaying for ACC2 Catalytic Activity

The activity of the ACC2 polypeptides of the present invention can bedetermined using a variety of ACC2 activity assays. Some representativeassays are described herein below.

A CO₂-fixation assay is an ACC assay that can be employed in the presentinvention. In this assay, [¹⁴C]—NaHCO₃, acetyl CoA, Mg-ATP, citrate andACC are incubated at 37° C.; the reaction mixture is quenched with acidat the end of the reaction, and subsequently heated to removebicarbonate as ¹⁴CO₂. Scintillant is then added and the acid-stablemalonyl CoA remaining in the vial is counted in a scintillation counter(see Waite & Wakil (1962) J. Biol. Chem. 237:2750-2757 and Tanabe et al.(1981) Methods Enzymol. 71 Pt C, 5-16).

The continuous ATP regeneration-coupled spectrophotometric assay isanother ACC2 assay that can be employed in the present invention. Inthis assay, the ADP generated in the ACC enzyme reaction is converted toATP by a pyruvate kinase/lactate dehydrogenase coupled enzyme system,and NADH disappearance is followed at 340 nm spectrophotometrically orfluorometrically (see Tanabe et al., (1981) Methods Enzymol. 71 Pt C,5-16). The ATP-regeneration system is very sensitive to the presence ofATPases.

Yet another form of ACC assay is an ACC/FAS coupled assay. In the ACCreaction, malonyl CoA is formed from acetyl CoA. Malonyl CoA can then beused as a substrate for FAS with NADPH as the cofactor. The reaction canbe monitored by the rate of utilization of NADPH spectrophotometrically(see Wakil et al., (1959) Biochim. Biophys. Acta 34:227-233).

Another method of assaying ACC2 enzymatic activity that can be employedin the present invention is described in U.S. Patent Application Ser.No. 60/558,015. In one embodiment of this assay, the method comprises:(a) contacting an enzyme mix comprising ACC and FAS, optionallycomprising one or more of bicarbonate, Mg, ATP, NADPH and an ACCeffector, with a solid support comprising a scintillant and a linkingmoiety; (b) incubating the enzyme mix with an acetyl CoA mix comprisingradiolabeled acetyl CoA, optionally comprising one or more ofbicarbonate, Mg, ATP, NADPH and an ACC effector, under suitable reactionconditions, for a desired incubation time; and (c) detectingscintillation signal, wherein scintillation signal is indicative of ACCcatalytic activity.

IX. Transgenic Animals

The preparation of a transgenic non-human animal that expresses anACC2-encoding sequence of the present invention is within the scope ofthe present invention. A preferred transgenic animal is a mouse.

Techniques for the preparation of transgenic animals are known in theart. Representative techniques are described in the literature and willbe known to those of ordinary skill in the art. Some representativetechniques are described, for example, in U.S. Pat. No. 5,489,742(transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396,5,625,125 and 5,648,061 (transgenic mice); U.S. Pat. No. 5,573,933(transgenic pigs); U.S. Pat. No. 5,162,215 (transgenic avian species)and U.S. Pat. No. 5,741,957 (transgenic bovine species).

In one method for the preparation of a transgenic mouse, clonedrecombinant or synthetic DNA sequences or DNA segments encoding a humanACC2 gene product are injected into fertilized mouse eggs. The injectedeggs are implanted in pseudo pregnant females and are grown to term toprovide transgenic mice whose cells express a human or rat ACC2 geneproduct.

X. Antibodies

In another aspect, the present invention relates to antibodies thatspecifically recognize the ACC2 proteins of the present invention. Suchantibodies can be employed in a range of applications. By way ofnon-limiting example, antibodies of the present invention can be used topurify, detect, and/or target the polypeptides of the present invention,including both in vitro and in vivo diagnostic and therapeutic methods.For example, antibodies can be employed in immunoassays forqualitatively and/or quantitatively measuring levels of the polypeptidesof the present invention in biological samples (see, e.g., Harlow &Lane., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2nd ed. (1988), incorporated by reference herein in itsentirety).

Antibodies of the present invention include, but are not limited to,polyclonal, monoclonal, monovalent, bispecific, heteroconjugate,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, encompasses immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

The terms “antibody” (Ab) and “monoclonal antibody” (Mab) encompassesintact molecules, as well as, antibody fragments (such as, for example,Fab and F(ab′)2 fragments) which are capable of specifically binding toa given protein. Fab and F(ab′)2 fragments lack the Fc fragment ofintact antibody, often clear more rapidly from the circulation of theanimal or plant, and may have less non-specific tissue binding than anintact antibody. Thus, in some situations these fragments are preferred,as well as the products of a Fab or other immunoglobulin expressionlibrary.

Antibodies of the present invention include chimeric, single chain, andhumanized antibodies. For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g. Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; Boulianne et al., Nature 312:643 (1984); and Neuberger etal., Nature 314:268 (1985), which are incorporated herein by referencein their entirety.

A humanized antibody, which is a form of chimeric antibody, comprises aportion of an antibody molecule from non-human species that binds thedesired antigen, and can comprise one or more complementaritydetermining regions (CDRs) from the non-human species, and a frameworkregion from a human immunoglobulin molecule. Often, framework residuesin the human framework region(s) will be substituted with thecorresponding residue from the CDR donor antibody to alter, and oftenimprove, antigen binding. These framework substitutions can beidentified by methods well known in the art, e.g. by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions (see, e.g.Riechmann et al., Nature 332:323 (1988)). Antibodies can be humanizedusing a variety of techniques known in the art including, for example,CDR-grafting (U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089),veneering or resurfacing (Padlan, Mol. Immunol. 28(4/5):489-498 (1991);Studnicka et al., Prot. Engineering 7(6):805-814 (1994); Roguska. etal., Proc. Natl. Acad. Sci. U.S.A. 91:969-973 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332). In some cases, a humanized antibodycomprises one or more amino acid residues introduced from a source thatis non-human. Methods of humanizing antibodies are known in the art; forexample, humanization can be performed essentially as described in Joneset al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-327(1988); and Verhoeyen et al., Science 239:1534-1536 (1988), namely bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies.

In general, a humanized antibody comprises substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin (Jones et al., Nature321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art, including phage display methods usingantibody libraries derived from human immunoglobulin sequences. Thetechniques of Cole et al., and Boerder et al., are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, New York (1985); andBoerner et al., J. Immunol. 147(1):86-95 (1991)).

Human antibodies can be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tohuman heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. The modified embryonic stem cells are thenexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies.

The transgenic mice can be immunized in the normal fashion with aselected antigen, e.g., all or a portion of an ACC2 polypeptide of thepresent invention. Monoclonal antibodies directed against the antigencan be obtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see, e.g., Lonberg & Huszar, Int. Rev.Immunol. 13:65-93 (1995).

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in Marks et al., Biotechnol.10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwildet al., Nature Biotechnol. 14:845-51 (1996); Neuberger, NatureBiotechnol. 14:826 (1996); and Lonberg & Huszer, Intern. Rev. Immunol.13:65-93 (1995).

In general, a humanized antibody comprises substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-humanimmunoglobulin, and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

The antibodies of the present invention can comprise monoclonalantibodies. Monoclonal antibodies can be prepared using hybridomamethods, such as those described by Kohler & Milstein, Nature, 256:495(1975) and Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, 2^(nd) ed. (1988); additional methods are knownto those of ordinary skill in the art. Other examples of methods whichmay be employed for producing monoclonal antibodies includes, but arenot limited to, the human B-cell hybridoma technique (Cole et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)), and theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies And CancerTherapy, Alan R. Liss, New York, pp. 77-96 (1985)). Such antibodies maybe of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing a mAb of this invention can becultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this a preferred method of production in some situations.

It is generally desirable that immortalized cell lines fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Generally, methods of preparing antibodies are known in the artand can be employed in the present invention to raise antibodies againstan ACC2 polypeptide of the present invention.

XI. Antisense and RNAi Methods

The present invention encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a coding strand of adouble-stranded cDNA molecule (i.e., a sense strand) encoding an ACC2protein of the present invention or a sequence that is complementary toan mRNA sequence. An antisense nucleic acid of the invention comprises asequence that is complementary to at least a portion of an RNAtranscript of a gene of interest. Absolute complementarity is notrequired. The ability to hybridize depends on both the degree ofcomplementarity and the length of the antisense nucleic acid Generally,the larger the hybridizing nucleic acid, the more base mismatches with apolynucleotide sequence of the present invention it can contain andstill form a stable multiplex structure (e.g., a duplex or triplex). Oneof ordinary skill in the art can readily ascertain a tolerable degree ofmismatch by employing standard procedures to determine the melting pointof the hybridized complex. Antisense nucleic acids are preferably atleast six nucleotides in length, and more preferably 6 to about 50nucleotides in length. In specific embodiments, an antisenseoligonucleotide comprises at least 10 nucleotides, at least 17nucleotides, at least 25 nucleotides or at least 50 nucleotides.

Antisense sequences of the invention can be chemically synthesized bystandard methods known in the art (see, e.g. Stein et al., Nucl. AcidsRes. 16:3209 (1988) and Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988), or by employing an automated DNA synthesizer, manyof which are commercially available. An antisense sequence of thepresent invention can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the multiplex formed between the antisense andsense nucleic acids, including, but not limited to, phosphorothioatederivatives and acridine substituted nucleotides.

Antisense sequences can also be prepared in vivo, again by employingtechniques known to those of ordinary skill in the art. For example, anantisense sequence can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest).

An antisense nucleic acid can be complementary to an entire codingstrand of the present invention, or to only a portion thereof, e.g., allor part of the protein coding region. An antisense sequence can also becomplementary to a noncoding region of the coding strand of a nucleotidesequence encoding an ACC2 polypeptide. For example, an antisensesequence can be complementary to the region surrounding the translationstart site of ACC2 mRNA. By employing the polynucleotide sequencesencoding the ACC2 polypeptides provided herein, an antisense sequence ofthe present invention can be readily designed based on standard basepairing rules.

The antisense sequences of the present invention can also be used intherapeutic applications to reduce or eliminate ACC2 activity in vivo.When used therapeutically, antisense sequences of the present inventionare typically administered to a subject or generated in situ such thatthey hybridize with or bind to cellular mRNA and/or genomic DNA encodinga GPCR-like protein to thereby inhibit expression of the protein, e.g.,by inhibiting transcription and/or translation. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. In other examples, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, antisense molecules can belinked to peptides or antibodies to form a complex that specificallybinds to receptors or antigens expressed on a selected cell surface.

An antisense sequence of the present invention can comprise DNA or RNAand can be used to control gene expression via the formation ofmultiplexes or via traditional antisense methodology. Antisensetechniques are discussed, for example, in Okano, J. Neurochem. 56:560(1991) and in Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton (1988). Triple helix formationoptimally results in a shut-off of RNA transcription from DNA, whileantisense RNA hybridization blocks translation of an mRNA molecule intopolypeptide. Both methods have previously been demonstrated to beeffective in model systems, and the information disclosed herein can beused to design antisense or triple helix polynucleotides in an effort totreat or prevent disease.

RNA interference (RNAi) reagents form another aspect of the presentinvention. RNAi is a process by which a target gene can be specificallysilenced. The RNAi process is activated when a double-stranded RNAmolecule of greater than about 19 duplex nucleotides (referred to hereininterchangeably as dsRNA and “RNAi reagent”) enters a cell, which causesthe degradation of not only the invading dsRNA molecule itself, but alsosingle-stranded RNAs of identical sequences, including endogenous mRNAs.As such, RNAi is a powerful tool in the development of highly specificRNA-based gene-silencing therapeutics, an aspect of the presentinvention. Thus, in one aspect of the present invention, RNAinterference (RNAi) methodologies can be employed to selective inhibitthe expression of a target gene in a vertebrate, which can form anelement of a therapeutic regimen.

RNAi reagents of the present invention can be obtained using any of anumber of techniques known to those of ordinary skill in the art.Generally, production of RNAi reagents can be carried out by chemicalsynthetic methods or by recombinant nucleic acid techniques. Methods ofpreparing a dsRNA are described, for example, in Ausubel et al., CurrentProtocols in Molecular Biology (Supplement 56), John Wiley & Sons, NewYork (2001); Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor (2001); and canbe employed in the present invention. For example, RNA can betranscribed from PCR products, followed by gel purification. Standardprocedures known in the art for in vitro transcription of RNA from PCRtemplates. For example, dsRNA can be synthesized using a PCR templateand the Ambion T7 MEGASCRIPT, or other similar, kit (Austin, Tex.); theRNA can be subsequently precipitated with LiCl and resuspended in abuffer solution.

An RNAi reagent of the present invention can be both partially orcompletely double-stranded. Generally, an RNAi reagent of the presentinvention encompasses fragments of at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 30, at least 35, at least 40, at least 45, and at least 50 or morenucleotides per strand. An RNAi reagent can also comprise 3′ overhangsof at least 1, at least 2, at least 3, or at least 4 nucleotides.Broadly, an RNAi reagent of the present invention can be of any lengthdesired by the user as long as the ability to inhibit target geneexpression is preserved.

An RNAi reagent of the present invention can include modifications tothe phosphate-sugar backbone or the nucleoside, e.g., to reducesusceptibility to cellular nucleases, improve bioavailability, improveformulation characteristics, and/or change other pharmacokineticproperties. For example, the phosphodiester linkages of natural RNA canbe modified to include at least one of an nitrogen or sulfur heteroatom.Other modifications that can be desirable under certain conditions willbe known to those of ordinary skill in the art. Modifications in RNAstructure can be tailored to allow specific genetic inhibition whileavoiding a general response to dsRNA.

An RNAi reagent of the present invention, can also be synthesized invitro (see, e.g., Fire et al., Nature 391:806-811 (1998); Montgomery etal., Proc. Natl. Acad Sci U.S.A. 95:15502-15507; Tabara et al., Science282:430-431 (1998)). Additionally, commercially available polynucleotidesynthesizers can be employed to prepare an RNAi reagent.

At least two ways can be employed to achieve siRNA-mediated genesilencing. First, siRNAs can be synthesized in vitro and introduced intocells to transiently suppress gene expression, for example as acomponent of a therapeutic regimen. Synthetic RNAi reagents provide aneasy and efficient way to achieve RNAi. Using synthetic 21 base pairduplexes, sequence specific gene silencing can be achieved in mammaliancells. These RNAi reagents can specifically suppress targeted genetranslation in mammalian cells without activation of DNA-dependentprotein kinase (PKR) by longer dsRNA, which may result in non-specificrepression of translation of many proteins.

Additionally, RNAi reagents can be expressed in vivo from vectors. Thisapproach can be used to stably express RNAi reagents in cells ortransgenic animals. In one embodiment, RNAi reagent expression vectorsare engineered to drive transcription from polymerase III (pol III)transcription units. The Pol III expression vectors can also be used tocreate transgenic mice that express siRNA.

In another embodiment, siRNAs can be expressed in a tissue-specificmanner. In this approach, long double-stranded RNAs (dsRNAs) are firstexpressed from a promoter (such as CMV (pol II)) in the nuclei ofselected cell lines or transgenic mice. The long dsRNAs are processedinto siRNAs in the nuclei (e.g., by Dicer). The siRNAs exit from thenuclei and mediate gene-specific silencing. A similar approach can beused in conjunction with tissue-specific (pol II) promoters to createtissue-specific knockdown mice.

An antisense sequence or RNAi reagent of the present invention can beadministered as part of a therapeutic regimen in which repression ofACC2 expression is desired. In this application, the antisense sequenceor RNAi reagent can be administered as a component of a bufferedsolution, or it can be administered as a component of a pharmaceuticalcomposition, as described herein.

XII. ACC2 Modulators and Screening Methods

The present invention broadly encompasses modulators of ACC2 and methodsof identifying such modulators. As used herein, the term “modulator”means a compound that can act as an agonist or an antagonist. An ACC2modulator can be employed in the various methods of the presentinvention, for example as a component of a therapeutic regimen. Suchmodulators can comprise for example, one, or a combination of, apolypeptide of variable length (including antibodies and fusionproteins) or a small molecule.

A modulator of the present invention can comprise any type of chemicalentity, such as a protein of any size, a small molecule or an antibody.Just as there is no limitation on whether a modulator augments orinhibits ACC2 or ACC2-mediated activity, there is no limitation on themechanism by which a modulator of ACC2 achieves such an effect. Forexample, a modulator might block a ligand from associating with an ACC2polypeptide. In another case, a modulator might inhibit an ACC2polypeptide from associating with another polypeptide. In yet anothercase, a modulator might facilitate the association of an ACC2polypeptide with another polypeptide expressed.

General methods of identifying modulators are known in the art and canbe employed in the present invention. Such methods will employ apolypeptide or polynucleotide sequence of the present invention (e.g.,SEQ ID NOs:11-14) as a target.

The ACC2 polypeptides and/or peptides of the present invention, orimmunogenic fragments or oligopeptides thereof, can be used forscreening therapeutic drugs or test compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaycan be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition of theformation of binding complexes between an ACC2 protein of the presentinvention and a test agent can be measured. Thus, the present inventionprovides a method for screening or assessing one or a plurality of testcompounds for their ability to specifically bind an ACC2 polypeptide, ora bindable peptide fragment thereof, of the present invention,comprising providing a plurality of compounds, combining an ACC2polypeptide, or a bindable peptide fragment thereof, with each of aplurality of test compounds for a time sufficient to allow binding undersuitable conditions and detecting binding of the ACC2 polypeptide orpeptide to each of the plurality of test compounds, thereby identifyingthe compounds that specifically bind to the ACC2 polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the ACC2polypeptides and/or peptides are provided in an aspect of the presentinvention and, in one embodiment, comprise combining a potential orcandidate compound or drug modulator of ACC2 biological activity with anACC2 polypeptide or peptide, for example, an ACC2 amino acid sequence asset forth in SEQ ID NOs:12 and 14, and measuring an effect of thecandidate compound or drug modulator on the biological activity of theACC2 polypeptide or peptide. Such measurable effects include, forexample, physical binding interaction; the ability to cleave a suitablesubstrate; effects on native and cloned ACC2-expressing cell line; andeffects of modulators or other ACC2-mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of an ACC2 polypeptide of the present invention comprisescombining a potential or test compound or drug modulator of ACC2biological activity with a host cell that expresses an ACC2 polypeptideand measuring an effect of the test compound or drug modulator on thebiological activity of the ACC2 polypeptide. The host cell can also becapable of being induced to express the ACC2 polypeptide, e.g., viainducible expression.

The physiological effects of a given test compound on an ACC2polypeptide can also be measured. Thus, cellular assays for particularACC2 modulators can comprise either direct measurement or quantificationof the physical biological activity of the ACC2 polypeptide, or they canbe measurement or quantification of a physiological effect. Such methodspreferably employ an ACC2 polypeptide as described herein, or anoverexpressed recombinant ACC2 polypeptide in suitable host cellscomprising an expression vector as described herein, wherein the ACC2polypeptide is expressed, overexpressed, or undergoes upregulatedexpression.

Another aspect of the present invention encompasses a method ofscreening for a compound that is capable of modulating the biologicalactivity of an ACC2 polypeptide and comprises providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a ACC2 polypeptide, or a functional peptide or portion thereof,determining the biological activity of the expressed ACC2 polypeptide inthe absence of a test compound; contacting the cell with the testcompound and determining the biological activity of the expressed ACC2polypeptide in the presence of the test compound. In such a method, adifference between the activity of the ACC2 polypeptide in the presenceof the test compound and in the absence of the test compound indicates amodulating effect of the compound.

Any chemical compound can be employed as a test compound in the assaysof the present invention. Compounds tested as ACC2 modulators can be anysmall chemical compound, or biological entity (e.g., protein, sugar,nucleic acid, lipid). Test compounds will typically be small chemicalmolecules. Generally, compounds employed as potential modulators can becapable of dissolution in aqueous or organic (e.g., DMSO-based)solutions. The assays of the present invention can be employed to screenlarge chemical libraries by automating the assay steps and providingcompounds from any convenient source. Assays can be run in parallel, forexample, in microtiter formats on microtiter plates in robotic assays.Test compounds can be purchased from a supplier or synthesized byappropriate methods known to those of ordinary skill in the art.

High throughput screening methodologies are suitable for the detectionof modulators of the ACC2 polynucleotides and polypeptides describedherein. Such high throughput screening methods can involve providing acombinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (e.g., ligand or modulator compounds).Such combinatorial chemical libraries or ligand libraries are thenscreened in one or more assays to identify those library members (e.g.,particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds so identified can serve as leadcompounds, or can themselves be used as potential or actualtherapeutics.

The present invention, therefore, provides methods of screening fordrugs or any other agents which affect activities mediated by the ACC2polypeptides of the present invention. In one embodiment, these methodscomprise contacting a test compound with a polypeptide of the presentinvention, or a fragment thereof, and assaying for the presence of acomplex between the agent and the polypeptide or a fragment thereof, thepresence of which can be determined using methods well known to those ofordinary skill in the art.

Thus, the use of the ACC2 polypeptides of the present invention, or thepolynucleotides encoding these polypeptides, to screen for moleculeswhich modify the activities of the ACC2 polypeptides of the presentinvention form an aspect of the present invention. One embodiment ofsuch a method comprises contacting an ACC2 polypeptide of the presentinvention with a test compound(s) suspected of having modulatory (e.g.,antagonist or agonist) activity, and then assaying the activity of thepolypeptide following binding.

In another aspect of the present invention, therapeutic compounds can bescreened by employing the an ACC2 polypeptide of the present invention,or binding fragments thereof, in any of a variety of drug screeningtechniques. The polypeptide or fragment employed in such a test can beaffixed to a solid support, expressed on a cell surface, free insolution, or located intracellularly. One method of drug screeningemploys eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the polypeptide orfragment. Drugs are screened against such transformed cells incompetitive binding assays. One can measure, for example, the formationof complexes between the agent being tested and a polypeptide of thepresent invention.

XIII. Diagnostic/Prognostic Assays and Kits

The present invention encompasses diagnostic/prognostic assays and kits.Such assays and kits can be employed to detect the presence, absenceand/or expression level of an ACC2 polypeptide of the present invention.The assays and kits of the present invention can also be employed toidentify or predict the presence of an adverse condition associated withACC2, such as obesity or diabetes, or the likelihood that a subject willacquire such a condition. Non-limiting examples diagnostic methods andkits that can be employed in the present invention are provided.

Increased or decreased expression of an ACC2 gene in a subject affectedwith a certain condition, such as obesity or diabetes, both of which areknown to be associated with ACC2, as compared to unaffected organismscan be assessed using the ACC2-encoding polynucleotides of the presentinvention. For example, altered expression, chromosomal rearrangement,or the presence of a mutation can be used as a diagnostic or prognosticmarker for the presence of or predisposition to diabetes or obesity.These diagnostic applications can employ an ACC2 polynucleotide of thepresent invention.

Thus, the present invention provides a diagnostic method useful for thediagnosis of a disorder or condition. In one embodiment, the methodinvolves measuring the expression level of an ACC2 polynucleotide of thepresent invention in cells, tissue or body fluid from an organism andcomparing the measured gene expression level with a standard level ofACC2 polynucleotide expression, whereby an increase or decrease in thegene expression level compared to the standard is indicative of adisorder.

As used herein, the phrase “measuring the expression level of apolynucleotide of the present invention” means making qualitative,quantitative and estimated measurements of (a) the degree to which anACC2 polypeptide of the present invention is expressed, or (b) the levelof the mRNA encoding an ACC2 polypeptide in a first biological sample,either directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing the polypeptidelevel in the first biological sample to the polypeptide level or mRNAlevel in a second biological sample). In one embodiment, the polypeptidelevel or mRNA level in the first biological sample is measured orestimated and compared to a standard polypeptide level or mRNA level,wherein the standard is taken from a second biological sample obtainedfrom an individual not having the disorder or determined not to have thedisorder by averaging levels from a population of organisms not having adisorder. Once a standard polypeptide level or mRNA level is known, itcan be used repeatedly as a standard for comparison.

The method(s) provided herein can be applied in a diagnostic methodand/or kits in which polynucleotides and/or polypeptides are attached toa solid support. In one exemplary method, the support may be a “genechip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832,5,874,219, and 5,856,174. Further, a gene chip comprising an ACC2polynucleotide of the present invention can be employed to identifypolymorphisms between reference ACC2 polynucleotide sequences, and ACC2polynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) can bebeneficial in identifying disease loci for an ACC2-associated disorder.

In addition to various detection and purification applications, theanti-ACC2 antibodies of the present invention can also be employed indiagnostic and prognostic applications. For example, such antibodies canbe employed to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g. to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance.

Continuing, labeled antibodies, and derivatives and analogs thereof,that specifically bind to an ACC2 polypeptide of the present inventioncan be used to detect, diagnose, or monitor diseases, disorders, and/orconditions associated with the aberrant expression and/or activity of anACC2 polypeptide of the present invention. Antibodies of the inventioncan be used to assay ACC2 levels in a biological sample using classicalimmunohistological methods known to those of ordinary skill in the art.Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA).

Examples of detectable labels include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of enzymes suitable foruse as a label include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of prosthetic groupcomplexes suitable for use as a label include streptavidin/biotin andavidin/biotin; examples of fluorescent materials suitable for use as alabel include umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin; an example of a luminescent material suitable for use asa label includes luminol; examples of bioluminescent materials suitablefor use as a label include luciferase, luciferin, and aequorin; andexamples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, or³H.

In one aspect, the present invention provides for the detection ofaberrant expression of an ACC2 polypeptide of the present invention,comprising (a) assaying the expression of the ACC2 polypeptide in cellsor body fluid of an individual using one or more antibodies specific tothe ACC2 polypeptide; and (b) comparing the level of gene expressionwith a standard gene expression level, wherein an increase or decreasein the assayed polypeptide gene expression level compared to thestandard expression level is indicative of aberrant expression.

The present invention also provides a diagnostic assay for diagnosing adisorder. In one embodiment the assay comprises (a) assaying theexpression of an ACC2 polypeptide of the present invention in cells orbody fluid of an individual using one or more antibodies specific to theACC2 polypeptide; and (b) comparing the level of gene expression with astandard gene expression level, whereby an increase or decrease in theassayed ACC2 polypeptide gene expression level compared to the standardexpression level is indicative of a particular disorder. With respect tocancer, the presence of a relatively high amount of transcript inbiopsied tissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer.

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with an ACC2 protein, ACC2nucleic acid expression, or ACC2 activity. Prognostic assays can be usedfor prognostic or predictive purposes to thereby prophylactically treatan individual prior to the onset of a disorder characterized by orassociated with an ACC2 protein, ACC2 nucleic acid expression, or ACC2activity.

In another aspect, the present invention provides a diagnostic method inwhich a test sample is obtained from a subject, and an ACC2 protein ornucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presenceof an ACC2 protein or nucleic acid is diagnostic for a subject having orat risk of developing a disease or disorder associated with aberrantACC2 expression or activity.

The present invention also provides a method of detecting geneticlesions or mutations in an ACC2 gene, thereby determining if a subjectwith the lesioned gene is at risk for an ACC2-related disorder. In oneembodiment, the method comprises detecting, in a biological sampleobtained from a subject, the presence or absence of a genetic mutationcharacterized by an alteration affecting the integrity of a geneencoding an ACC2-protein, or the misexpression of an ACC2 gene. Forexample, such genetic mutations can be detected by determining thepresence or absence of at least one of: (1) a deletion of one or morenucleotides from an ACC2 gene; (2) an addition of one or morenucleotides to an ACC2 gene; (3) a substitution of one or morenucleotides in an ACC2 gene; (4) a chromosomal rearrangement of an ACC2gene; (5) an alteration in the level of a messenger RNA transcript of anACC2 gene; (6) an aberrant modification of an ACC2 gene, such as of themethylation pattern of the genomic DNA; (7) the presence of anon-wild-type splicing pattern of a messenger RNA transcript of an ACC2gene; (8) an undesirable level of an ACC2 protein; (9) an allelic lossof an ACC2-like gene; and (10) an inappropriate post-translationalmodification of a GPCR-like-protein. There are a large number of assaytechniques known in the art that can be used for detecting mutations inan ACC2 gene

The present invention also encompasses kits for detecting the presenceof ACC2 proteins in a biological sample (a test sample). Such kits canbe used to determine if a subject is suffering from or is at increasedrisk of developing a disorder associated with aberrant expression ofACC2 protein (e.g., diabetes). For example, a kit can comprise a labeledcompound or agent capable of detecting an ACC2 protein or mRNA in abiological sample and means for determining the amount of an ACC2protein in the sample (e.g., an anti-ACC2 antibody or an oligonucleotideprobe that binds to DNA encoding an ACC2 protein, e.g. SEQ ID NOs:12 and14). A kit can also include instructions for interpreting observedresults and/or steps for performing the assay.

For antibody-based kits, a kit can comprise, for example: (1) a firstantibody, optionally attached to a solid support, that binds an ACC2polypeptide of the present invention; and, optionally, (2) a second,different antibody that binds an ACC2 polypeptide of the presentinvention or the first antibody and is conjugated to a detectable label.For oligonucleotide-based kits, a kit can comprise, for example: (1) anoligonucleotide, optionally a detectably labeled oligonucleotide, thathybridizes to an ACC2 polynucleotide sequence of the present invention,or (2) a pair of primers useful for amplifying an ACC2 polynucleotide.

Depending on the nature of the kit, a kit of the present invention canalso comprise other components, such as a buffering agent, apreservative, or a protein stabilizing agent. The kit can also comprisecomponents that facilitate the detection of the label. The kit can alsocontain a control sample or a series of control samples that can beassayed and compared to the test sample contained. A diagnostic kit ofthe present invention can also comprise instructions to assist the userin performing the diagnostic test and/or interpreting the results of thediagnostic test.

XIV. Pharmaceutical Compositions

An ACC2 polypeptide of the present invention, with or without atherapeutic agent conjugated to it, can be administered alone or incombination with a another biologically active moiety, including a smallmolecule, and can be used as a therapeutic. Additionally, modulators ofthe ACC2 polypeptides of the present invention form another aspect ofthe invention.

The present invention provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an ACC2polypeptide or an ACC2 modulator of the present invention, and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water and water-basedformulations are desirable carriers when the pharmaceutical compositionis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, (Gennaro, ed.)20th ed., Mack Publishing, Easton, Pa., USA (2000). Such compositionswill contain a therapeutically effective amount of the modulator, forexample in purified form, together with a suitable amount of carrier soas to provide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In one embodiment, a composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherea composition is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where a composition is administered by injection, an ampule ofsterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The amount of ACC2 polypeptide or ACC2 modulator that will be effectivein the treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of an ACC2polypeptide of the present invention can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the nature of the disease or disorder, and can bedecided according to the judgment of the practitioner and each subject'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

A pharmaceutical composition can be administered in conjunction with apharmaceutically acceptable carrier, diluent, or excipient, to achieveany of the above-described therapeutic uses and effects. Suchpharmaceutical compositions can comprise agonists, antagonists,activators or inhibitors. The compositions can be administered alone, orin combination with at least one other agent or reagent, such as astabilizing compound, which can be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions can beadministered to a patient alone, or in combination with other agents,drugs, hormones, or biological response modifiers.

The pharmaceutical compositions for use in the present invention can beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, orrectal means.

In addition to the active ingredients, the pharmaceutical compositionscan contain pharmaceutically acceptable/physiologically suitablecarriers or excipients comprising auxiliaries which facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Further details on techniques for formulation andadministration are provided in Remington's Pharmaceutical Sciences,(Gennaro, ed.) 20th ed., Mack Publishing, Easton, Pa., USA (2000).

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the present invention.Optionally a notice can be associated with such container(s) in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. Such a notice can also provide guidance on how to usethe pack or kit.

XV. Method of Increasing ACC2 Enzymatic Activity

In yet another aspect of the present invention, a method of increasingthe activity of a human ACC2 polypeptide is disclosed. In oneembodiment, the method comprises generating an enhanced ACC2 polypeptidecomprising (a) a phenylalanine residue at position 254, (b) a glutamineresidue at position 346, (c) a threonine residue at position 565, (d) anasparagine at position 841, (e) a valine residue at position 1103, (f) acysteine residue at position 1259, (g) an alanine residue at position1526, and (h) an isoleucine residue at position 1717, wherein the humanACC2 polypeptide does not comprise SEQ ID NO:12 and wherein the enhancedACC2 polypeptide has an enzymatic activity level that is greater thanthe enzymatic activity level of an ACC2 polypeptide that does notcontain the indicated residues at the indicated positions.

The present inventors have identified eight amino acids which, whenmutated to the residues described herein, impart increased activity toACC2. Although the ACC2 sequences of the present invention include pointmutations differentiating the ACC2 sequences of the present inventionfrom the published sequences, this group of mutations includes a coregroup of eight mutations, which are believed to affect the activity ofthe mutated enzyme. More particularly, when the noted residues of anACC2 sequence other than that of SEQ ID NO:12 are replaced with (a) F atposition 254, (b) Q at position 346, (c) T at position 565, (d) N atposition 841, (e) V at position 1103, (f) C at position 1259, (g) A atposition 1526, and (h) I at position 1717, a concurrent >200-foldincrease in activity is observed.

Consistent with the observations disclosed herein (see, e.g., Example9), a method of increasing ACC2 enzymatic activity is provided. In oneembodiment the method comprises introducing eight point mutations intoan ACC2 sequence other than the sequence of SEQ ID NO:12. The mutationsintroduced are (a) F at position 254, (b) Q at position 346, (c) T atposition 565, (d) N at position 841, (e) V at position 1103, (f) C atposition 1259, (g) A at position 1526, and (h) I at position 1717.Additional mutations can, but need not, be introduced.

It is possible that one or more residues may be present in a publishedACC2 sequence at the noted position(s). In this case, less than theeight mutations can be introduced, with the proviso that the final formof the mutated ACC2 sequence comprises the noted eight mutations. Aftermutations have been made, the activity of the resultant mutant can bedetermined as described herein and can be compared to an activitymeasurement made before any mutations were introduced.

The ACC2 sequence can comprise any known ACC2 sequence, such as the ACC2sequences of SEQ ID NOs:2, 4 or 6. When there is a residue other thanone of the specified eight residues at the corresponding position in theACC2 sequence, a point mutation can be introduced into the ACC2 sequenceto conform the residues and positions with the eight residue/positioncombinations indicated herein. Such mutations can be introduced usingstandard methodology, such as that provided herein.

EXAMPLES

The following Examples have been included to illustrate preferred modesof the invention. Certain aspects of the following Examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These Examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following Examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

Example 1 Identification of Human ACC2 Amino Acid Sequence

To identify the wild type human ACC2 amino acid sequences, the followingthree amino acid sequences were compared: 1) the sequence ofpYES-human-ACC2 (purchased from Dr. Ki-Han Kim, Purdue University), 2)the coding region of human ACC2 predicted by human genomic contigAC007637 (defined by 12q24 BAC RCPI11-443D10; Roswell Park CancerInstitute Human BAC Library, complete sequence), and 3) Genbank ACC2sequence (NM_(—)001093; Ha et al., (1996) Proc. Natl. Acad. Sci. USA93:11466-11470). The multiple sequence alignment was performed withClustalW algorithm in VectorNTI program. The result of the comparisonrevealed that there are multiple discrepancies among these threesequences. Specifically, in amino acid sequences predicted by genomiccontig AC007637, the discrepancies are C at position 9, G at position347, G at positions of 349-352, V at position 2141; in amino acidsequences described in Genbank Accession No. NM_(—)001093, thediscrepancies are H at position 111, V at position 127, S at position450, R at position 614, K at position 656, V at position 671, KI atposition 742-743, K at position 799, A at position 1025, A at position1064, A at position 1480, G at position 1547, A at position 1821, RPMRat position 2194-2197, E at position 2242; in amino acid sequences aspredicted by pYES-human-ACC2, the discrepancies are Y at position 254, Rat position 345, A at position 565, Y at position 841, A at position1103, R at position 1259, V at position 1526, V at position 1717. Theconsensus of amino acid sequences among pYES-human-ACC2, GenbankAccession No. NM_(—)001093 and human genome contig AC007637 is definedas the wild type human ACC2 amino acid sequence (SEQ ID NO:12).Specifically the amino acids need to be R at position 9, P at position111, A at position 127, F at position 254, Q at position 345, V atposition 347, AGWG at positions of 349-352, P at position 450, T atposition 565, H at position 614, E at position 656, E at position 671,ET at position 742-743, E at position 799, N at position 841, V atposition 1025, V at position 1064, V at position 1103, C at position1259, R at position 1480, A at position 1526, R at position 1547, I atposition 1717, G at position 1821, I at position 2141, PPYA at position2194-2197, K at position 2242.

Example 2 Identification of Rat ACC2 Amino Acid Sequence

Initially the published sequences for both genes were used to designprimers to amplify overlapping fragments. Two different clones for eachfragment were sequenced and aligned using ClustalW algorithm inVectorNTI program with the published sequences (SEQ ID NOs:7 and 9;GenBank Accession Nos. NM_(—)053922 and AB004329, respectively). Using aconsensus sequence built from this comparison, the consensus translationwas then aligned with the published rat ACC2 sequence (SEQ ID NO: 9,FIGS. 3 and 4) and the sequence of human ACC2 (SEQ ID NO: 2) wasemployed to guide decisions surrounding ambiguous regions, providing arefined consensus sequence. This refined consensus sequence was thencompared to the rat genomic ACC2 region and provided an identical match(FIG. 5).

Materials for Examples 3 to 5

Expression vectors pBlueBac4.5/V5-His, pcDNA4/V5-His a Bac-N-BlueTransfection kit, and monoclonal anti-V5 antibody was obtained fromInvitrogen; goat anti-mouse horseradish peroxidase (HRP)-conjugatedantibody was obtained from BioRad; streptavidin conjugated with HRP wasobtained from Pierce; COMPLETE® protease inhibitor cocktail tablets andFuGene 6 transfection reagent was obtained from Roche MolecularBiochemicals; TALON™ resin was obtained from BD Biosciences;oligonucleotide primers were obtained from Sigma-Genosys; a LA-PCR kitand a ligation kit was obtained from Panvera Corporation; a QUIKCHANGE®multi site-directed mutagenesis kit was obtained from Stratagene; humanembryonic kidney (HEK) 293 cells and insect Sf9 cells were obtained fromATCC; [¹⁴C]—NaHCO₃ (45 mCi/mmol, 1 mCi/m) was obtained from NEN LifeScience Products, Inc. C-Myc peptide (Ac-EQKLISEEDL-OH; SEQ ID NO:16)was synthesized.

Example 3 Construction of Human ACC2 Expression Vectors

To construct pBlueBac-human-ACC2, a ˜7.5 kb fragment of a Kpn I/Xho Idouble digestion of pYES-human-ACC2 (purchased from Dr. Ki-Han Kim,Purdue University; SEQ ID NO: 5) was ligated with a pBlueBac4.5 vectordigested with same set of restriction enzymes.

To create a V5-His tag at human ACC2 COOH-terminus, a PCR reaction wascarried out using the following set of primers: forward:5′-TCCTGTATTGGCGTCTGCGCCGC-3′ (SEQ ID NO:17), reverse:5′-CGAATTCACGGTGGAGGCCGGGCTGTC-3′ (SEQ ID NO:18), and withpYES-human-ACC2 as the template. The resultant ˜490 bp product wasdigested with Esp3 I and EcoR I which is ligated with pBlueBac4.5/V5-Histhat was digested with Esp3 I and EcoR I. The resultant plasmid wasdesignated pBlueBac-human-ACC2-V5-His.

To construct mammalian expression construct, pBlueBac-human-ACC2-V5-Hiswas digested with Kpn I and Age I. The resultant ˜7.5 kb fragment wasligated with pcDNA4/V5-His that was digested with the same set ofenzymes. The resulting plasmid was designated pcDNA-human-ACC2-V5-His.

To delete the NH₂-terminus 148 amino acid of human ACC2, a PCR reactionwas carried out using the following set of primers: forward:5′-GGCCGAAGCCGGTACCGCCATGGGCAAAGAAGACAAGAAGCAGGCAAACATCAAGAGGCAGCTG-3′,(SEQ ID NO:19), reverse: 5′-CGTCTGGGCGACAACGGTGGA-3′ (SEQ ID NO:20), andwith pcDNA-human-ACC2-V5-His as the template. The PCR product wasdigested with ACC65 I and Sfi I and ligated with the ˜12.5 kb digestionproduct of pcDNA-human-ACC2-V5-His with the same set of enzymes. Theresultant plasmid was designated pcDNA-human-ACC2-Delta148-V5-His.

To insert myc tag between V5- and His-tags at the COOH terminus, a PCRreaction was carried out using the following set of primers: forward:5′-TCCTGTATTGGCGTCTGCGCCGC-3′ (SEQ ID NO:21), reverse:5′-GGCCGAAGCCACCGGTGCCCAGATCCTCTTCTGAGATGAGTTTTTGTTCGCCCGTAGAATCGAGACCGAGGAGAG-3′(SEQ ID NO:22), and with pcDNA-human-ACC2-Delta-V5-His as the template.The PCR product was digested with Esp3 I and with Age I, which wasligated with the ˜14 kb digestion product ofpcDNA-human-ACC2-Delta148-V5-His generated by digestion with the sameset of enzymes. The resultant plasmid was designatedpcDNA-human-ACC2-Delta148-V5-Myc-His.

To conduct site-directed mutagenesis in order to create a version thatcontains all the amino acids as predicted by wild type sequence (SEQ IDNO:12), two intermediate plasmids were constructed. First,pBlueScrip-ACC2-N and pBlueScript-ACC2-C were constructed.pBlueScrip-ACC2-N was constructed by ligation of 3.4 kb ACC65 I/Sac IIdigestion product of pcDNA-human-ACC2-Delta148-V5-Myc-His with ˜3.0 kbACC65 I/Sac II digestion product of pBlueScript II. pBlueScript-ACC2-Cwas constructed by ligation of the ˜4.0 kb Sac II/Pme I digestionproduct of pcDNA-human-ACC2-Delta148-V5-Myc-His with ˜3.0 kb Sac II/PmeI digestion product of pBlueScript II.

In order to change the discrepancies found in pYes-human-ACC2 thatreside in the 5′-3.4 kb portion, mutagenesis was conducted usingpBlueScript-ACC2-N as the template, using the following primers: (SEQ IDNO:23) 5′-CGATCCCCCCCAAAGCGTGTGACAAA-3′, (SEQ ID NO:24)5′-CCACACCGCCTGCACGGGGATTC-3′, (SEQ ID NO:25)5′-GAGGTATTCCACTGTCCCTGCACTCAC-3′v, (SEQ ID NO:26)5′-CGATGTGGCAGCCATTCATGATGAGAACG-3′, and (SEQ ID NO:27)5′-TGTTGATGAAGAAGACCTCTCGATCAGCCT-3′,using a QUIKCHANGE multi site-directed mutagenesis kit according to themanufacturer's instructions. Sequencing experiments were conducted toidentify clones that bear all the desired changes without introducingundesired changes. The resulting clone was designatedpBlueScript-ACC2-N-WT.

In order to change the discrepancies found in pYes-human-ACC2 thatresides in the 3′-4.0 kb portion, mutagenesis was conducted usingpBlueScript-ACC2-C as the template, using the following primers:

5′-GTTCTCGGGGCAGAACTGGTGGC-3′ (SEQ ID NO:28),

5′-CCTTCACCTTGGCAGCACCCAGGTAAAG-3′ (SEQ ID NO:29), and

5′-GGGAAGTCATAGATGTAGGTGGTTCCC-3′ (SEQ ID NO:30),

using QUIKCHANGE multi site-directed mutagenesis kit according to themanufacturer's instructions. Sequencing experiments were conducted toidentify clones that bear all the desired changes without introducingundesired changes. The resulting clone was designatedpBlueScript-ACC2-C-WT.

To reconstruct the expression vector containing all the wild type aminoacid sequences, a ligation was conducted to join the following threecomponents: the ACC65 I/Age I digestion product of pcDNA4/V5-His, the˜3.4 kb ACC65 I/Sac II digestion product of pBlueScript-ACC2-N-WT, andthe ˜4.0 kb Sac I/Age I digestion product of pBlueScript-ACC2-C-WT. Theresulting plasmid was designated pcDNA-human-ACC2-Delta148-V5-Myc-His-WT.

To construct a baculoviral vector comprising the final version humanACC2, the Kpn I/Age I digestion product of pBlueBac4.5/V5-His and theKpn I/Age I digestion product of pcDNA-human-ACC2-Delta148-V5-Myc-His-WTwere ligated. The resultant plasmid was designated aspBlueBac-human-ACC2-Delta148-V5-Myc-His-WT.

Example 4 Purification of Recombinant Human ACC2 by Anti-myc AffinityColumn

Affinity chromatography using c-Myc-5 monoclonal IgG column and wascarried out essentially according to Hillman et al (Hillman et al.,(2001) Protein Expression and Purification 23:359-368), with severalmodifications. Pellets of HEK-293 cells or Sf9 cells that were eithertransiently transfected with a human ACC2 expression vector or infectedwith a recombinant human ACC2 baculovirus were lysed in 5× cell pelletvolume of Buffer A (225 mM mannitol, 75 mM sucrose, 10 mM Tris/HCl. pH7.5, 0.05 mM EDTA, 1× complete protease inhibitor cocktail, 0.5 mM PMSF)with sonication for 10 seconds. The broken cells were centrifuged for 10minutes at 2000×g. NaCl concentration of supernatant was raised to ˜500mM with addition of 1/10 volume of 5 M NaCl. The cell lysates were thenfurther centrifuged for 30 minutes at 10,000×g at 4° C. The supernatantsof the second centrifugation were incubated with 3 ml resin coupled withc-Myc-5 IgG (density: 3 mg IgG/ml resin), which was pre-equilibratedwith Buffer B (100 mM Tris/HCl, pH 7.5, 0.5 M NaCl, 1 mM EDTA, 10%glycerol) overnight at 4° C. The incubated resin was then packed in acolumn and washed with Buffer B extensively (˜200 ml) until O.D.280reached baseline. After the wash, an aliquot of 1 ml Buffer C (Buffer Bcontaining 1 mM myc peptide) was carefully applied to the column. OnceBuffer C completely entered the column bed, column was plugged andincubated for 15 minutes at 4° C. After the incubation, the eluate wascollected via gravity. This elution procedure was repeated twelve times.The elution fractions were then subjected to analyses by coomassiestain, immunoblot and ACC enzymatic assays. Peak fractions were pooledand stored at −80° C. for further study.

Example 5 ACC Enzyme Activity Assay

ACC enzymatic assays were carried out essentially according to Tanabe etal. (Tanabe et al. (1981) Methods Enzymol. 71 Pt C, 5-16) withmodifications. In 7 ml glass scintillation vials, either fractions ofrecombinant human ACC2 eluted from the affinity column or ˜0.25 μg ofpooled purified ACC2 were mixed with 120 μl Buffer D (50 mM Hepes, pH7.5, 10 mM MgCl2, 10 mM tripotassium citrate, 0.1 mM DTT, 100 μg/ml BSA)that contains either 4 mM ATP, 250 μM acetyl CoA or at variousconcentrations as indicated in the figures. Aliquots of 30 μl of 25 mMKH[¹⁴C]O₃ (specific activity 1.3 μCi/μmol, final KH[¹⁴C]O₃,concentration 5 mM) was then added to the mixture to initiate thereaction which was carried out for 10 min at 37° C. At the end, thereactions were quenched with 50 μl 2 N HCl and the vials were heated for2 hours at 80° C. to remove excess bicarbonate as ¹⁴CO₂. Scintillant wasthen added and the acid-stable malonyl CoA remaining in the vial wascounted in a scintillation counter. ACC specific activities wereexpressed as nmol/min/mg protein.

Example 6 Immunoblot Analyses of Insect Sf9 Cells Expressing Human ACC2

FIG. 1A is a schematic diagram depicting the primary structure of theform of human ACC2 that is expressed in one aspect of the presentinvention. In order to increase the solubility of recombinant humanACC2, the first 27 hydrophobic amino acids and the following stretch ofamino acids (from 27 to 148) were deleted. This stretch of sequence hasbeen shown to facilitate the attachment of ACC2 with mitochondria(Abu-Elheiga et al., (2000) Proc. Nat. Acad. Sci. USA 97:1444) Sincethese sequences are not present in ACC1 enzyme (Ha et al., (1996) Proc.Natl. Acad. Sc. USA 93:11466-11470), it was deemed plausible that theyare not essential for the catalytic activity. It was also predicted thatdeleting the mitochondria attachment sequence would also minimizedocking too much over-expressed recombinant protein to the mitochondriaand thereby prevent a potential detriment to the host cells.

In order to facilitate the identification and purification of therecombinant enzyme, three consecutive tags were fused to theCOOH-terminus end of the enzyme, namely V5 and myc epitope-tags, and a6×His tag.

To ensure the fidelity of coding sequence in the recombinant human ACC2,the amino acid sequences of pYES-human-ACC2, the coding region of humanACC2 predicted by human genomic contig AC007637 and Genbank ACC2sequence (NM_(—)001093) (Ha et al. (1996) Proc. Natl. Acad. Sci. USA93:11466-11470) were compared. The result of the comparison revealedthat there are multiple discrepancies among these three sequences.Specifically, in amino acid sequences predicted by genomic contigAC007637, the discrepancies are C at position 9, G at position 347, G atpositions of 349-352, V at position 2141; in amino acid sequencesdescribed in Genbank Accession No. NM_(—)001093, the discrepancies are Hat position 111, V at position 127, S at position 450, R at position614, K at position 656, V at position 671, KI at position 742-743, K atposition 799, A at position 1025, A at position 1064, A at position1480, G at position 1547, A at position 1821, RPMR at position2194-2197, E at position 2242; in amino acid sequences as predicted bypYES-human-ACC2, the discrepancies are Y at position 254, R at position345, A at position 565, Y at position 841, A at position 1103, R atposition 1259, V at position 1526, V at position 1717. The consensus ofamino acid sequences among pYES-human-ACC2, Genbank Accession No.NM_(—)001093 and human genome contig AC007637 is defined as the wildtype human ACC2 amino acid sequence (SEQ ID NO:12). Specifically theamino acids need to be R at position 9, P at position 111, A at position127, F at position 254, Q at position 345, V at position 347, AGWG atpositions of 349-352, P at position 450, T at position 565, H atposition 614, E at position 656, E at position 671, ET at position742-743, E at position 799, N at position 841, V at position 1025, V atposition 1064, V at position 1103, C at position 1259, R at position1480, A at position 1526, R at position 1547, I at position 1717, G atposition 1821, I at position 2141, PPYA at position 2194-2197, K atposition 2242. The discrepancies in the human Genomic contig wereattributed to the presence of a sequencing error or polymorphism, andthe discrepancies in pYES-human-ACC2 and Genbank Accession No.NM_(—)001093 were attributed to the mutations possibly introduced by aPCR reaction during the cloning (Ha et al. (1996) Proc. Natl. Acad. Sci.USA 93:11466-11470).

In pYES-human-ACC2, there were eight single amino acid discrepanciesidentified, as compared to the designated wild type human ACC2 sequence.The discrepancies are distributed throughout the entire coding regionand correspond to the following mutations: F254Y, Q345R, T565A, N841Y,V1103A, C1259R, V1526A, I1711V At the nucleotide level, in all cases,single nucleotide changes were identified. In order to test thepossibility that these eight point mutations might change the protein'sstability or, alternatively, might change ACC enzyme activity, theseamino acids were systematically changed to the wild type sequence. FIG.6A depicts the number and location of the identified discrepancies.

Activity assays indicated that these eight point mutations decrease theactivity of the protein.

FIG. 6B demonstrates ACC2 protein expression levels, which weregenerated using immunoblot analyses. Probed with anti-V5 IgG antibodies(V5 is a common epitope present in both ACC2Mt (containing the originaleight mutations found in pYES-human-ACC2) and ACC2WT)), it was foundthat ACC2WT is more stable than ACC2Mt. This result was reproduced whenlysates were probed with Streptavidin-conjugated-with-HRP, which detectsthe biotin group on ACC. Further, there is a detectable signal inMock-infected Sf9 cells at the same molecular mass, indicating there issubstantial endogenous insect cell ACC enzyme present (FIG. 6B).

Example 7 Performance of Recombinant Human ACC2 on Monomeric AvidinColumn

Biotinylation of ACC is an indispensable protein modification for itsenzymatic function. To investigate the level of biotinylation of therecombinant human ACC2, lysates derived from cells expressing human ACC2were loaded on monomeric avidin column. The column was washed and wasthen eluted with 0.2 mM biotin for 3 hours (Elu1, lane 3), which wasfollowed by another overnight elution (Elu2, lane 4). Aliquots from theflow-through from the loading step (FT), two steps of elution, andmaterials remained in the column after two steps of biotin-elution(eluted by boiling in SDS loading buffer) were quantitatively loaded onSDS-PAGE and blot-analyzed with anti-V5 IgG. Almost no recombinant humanACC2 was detected in the flow-through fraction, whereas there was aquantitative recovery for human ACC2 bound to the avidin column. Thisindicates that nearly all the recombinant human ACC2 is properlybiotinylated (FIG. 7).

Example 8 Performance of Human ACC2 on TALON Resin

The observation that the recombinant human ACC2 cannot be eluted from amonomeric avidin column via competition with a high concentration ofbiotin suggests that in some situations this may not be a preferredmethod of purifying the recombinant human ACC2 of the present invention.Additionally, multiple attempts to purify that recombinant ACC2 enzymeusing conventional protein purification methods (including ammoniumsulfate precipitation, gel-filtration, ion-exchange chromatography) didnot provide the desired level of separation of these two types of ACCenzymes. Lastly, the observation that there is endogenous insect ACCenzyme in the host cells (FIG. 6B, lane 4), suggested that it might beadvantageous to employ an affinity tag that is capable ofdifferentiating the recombinant enzyme from the endogenous ACC.

The first affinity tag that was employed was TALON resin (Clontech, PaloAlto, Calif.), which can be used to purify recombinant poly-His-taggedproteins (Bush et al., (1991) J. Biol. Chem. 266:13811-13814). FIG. 8depicts a comparison of binding for the total lysates and recombinanthuman ACC2. The results suggest that there is an amount of non-specificbinding of host cell proteins to the TALON resin, particularly thoseproteins with high molecular mass (FIG. 8, lanes 1 to 4). Probingspecifically for human ACC2 indicates a loss of signal in the Eluatefraction as compared with the Load fraction (FIG. 8, lanes 6 and 8). ACCactivity measurement revealed that there is a small increase in ACCspecific activity before and after the sample was applied to the TALONcolumn. These results indicated that a 6×His-tag might not be desirablein all situations for the isolation of recombinant human ACC2 from thehost cell lysates.

Example 9 Purification of Recombinant Human ACC2 with Anti-Myc-IgG

It was hypothesized that a preferred affinity column for purifying humanrecombinant human ACC2 would have a high enough affinity to absorb theprotein (e.g., higher than Kd ˜10⁻³ M, the affinity reported by themanufacturer for the TALON-PolyHis resin) and to allow stringent washingcondition, yet have a low enough affinity for the protein to be eluted(e.g., smaller than Kd of ˜10⁻¹⁵ M, the avidin-biotin affinity; Hilleret al. (1987) Biochem. J. 248:167-171). Antibody-antigen interactions,for example, fall into this category. An anti-Myc-IgG antibody wastherefore selected for investigation. c-Myc-5 IgG has been extensivelycharacterized (see, e.g. Hillman et al., (2001) Protein Expres. Purif.23:359-368).

FIGS. 9A and 9B depict the purification of human ACC2 using a c-Myc-5IgG column. Total crude cell cytosolic lysates derived from 1 liter ofhuman ACC2 virus infected Sf9 cells were loaded on a 3-ml c-Myc-5 IgGcolumn by overnight incubation. The bound protein was then extensivewashed with high salt buffer (0.5 M NaCl). The most tightly boundproteins were then eluted with 1 mM myc peptide (Ac-EQKLISEEDL-OH; SEQID NO:16) in 10 steps. The protein peak of the eluate resided atfraction 4 and 5 as a single ˜250 kDa protein band (FIG. 9A). Thisprotein was recognized by anti-V5 and Streptavidin-conjugated-with-HRPin blot analyses, indicating that the purified band is recombinant humanACC2. In a parallel ACC enzymatic measurement, ACC activity peaks werefound in the same fractions as those identified in the coomassie stainassay (compare FIGS. 9A and 9B).

The active fractions were then pooled. A quantitative ACC enzyme assayindicated that the pooled enzyme has a specific activity of 500nmol/min/mg. The yield from 1 liter Sf9 cell culture was ˜2 mg protein.The recovery of total activity was 80%.

In order to address whether human-ACC2-Mt is different at enzymaticlevel as compared with human-ACC2-WT, human-ACC2-Mt was purified in thesame way as described above. In addition to the lower yield, in ACCenzyme assays human-ACC2-Mt was not observed to contain measurableactivity, indicating that one or several amino acids that wereidentified as discrepant between pYES-human-ACC2 and the wild type ACC2sequence is critical for ACC enzyme activity.

Example 10 Determination of Kinetic Parameters for Recombinant HumanACC2

In order to detect recombinant human ACC2 activity in one in vitro assay(see, e.g. U.S. Patent Application Ser. No. 60/558,015, incorporatedherein by reference), acetyl CoA, ATP, bicarbonate are preferablypresent. It was found that in the absence of any one of these compounds,no activity was detected. In addition, the presence of the knowneffector citrate was found to be required for the detection of ACC2activity in the assay employed. The K_(m) for the substrates, acetylCoA, ATP and bicarbonate and the K_(act) for the effector citrate weredetermined by assaying ACC activity at various concentrations of onereagent and saturating concentrations of all the others at 37° C.

FIG. 10 comprises plots depicting the concentration dependence of acetylCoA, ATP, bicarbonate and citrate. Table 1 summarizes the K_(m) andK_(act) value for the recombinant human ACC2 as compared with literaturevalues of rat ACC enzymes. TABLE 1 Kinetic Parameters of RecombinantHuman ACC2 Recombinant Literature Values* Human ACC2 Rat ACC1 Rat ACC2Km Acetyl CoA (uM) 23 22 32 ATP (uM) 270 110 58 HCO₃ ⁻(mM) 5 2.7 2.3Kact Citrate (mM) 1.8 3 2.1(*literature value from Trumble et al., (1995) Eur. J. Biochem. 231:192-198)

Example 11 Effect of Known Inhibitors for Recombinant Human ACC2

FIG. 11 comprises two plots depicting the concentration dependentinhibition of recombinant human ACC2 by known inhibitors such aspalmitoyl CoA and malonyl CoA. The IC₅₀ for these two agents wasdetermined to be 4.4 μM and 26.7 μM for palmitoyl CoA and malonyl CoA,respectively. For comparison, the literature IC₅₀ values of palmitoylCoA and malonyl CoA for rat ACC2 enzyme are 2.2 μM and 10.6 μM (Trumbleet al., (1995) Eur. J. Biochem. 231, 192-198).

REFERENCES

The references cited herein are incorporated herein by reference to theextent that they supplement, explain, provide a background for or teachmethodology, techniques and/or compositions employed herein. All citedpatents, including patent applications, and publications referred to inthis application are herein expressly incorporated by reference. Alsoexpressly incorporated herein by reference are the contents of allcitations of GenBank accession numbers, LocusID, and other computerdatabase listings, as well as the contents of any Sequence Listingassociated herewith.

While the invention has been described in connection with specificembodiments, it will be understood that the invention encompassesfurther modifications including variations, uses, and adaptations of theinvention that follow the principles of the invention. Furthermore, theforegoing description is for purposes of illustration.

1-7. (canceled)
 8. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) a polypeptidecomprising SEQ ID NO:13; (b) a polypeptide comprising amino acids 2 to2458 of SEQ ID NO:13, wherein amino acids 2 to 2458 comprise apolypeptide of SEQ ID NO:13 minus the start methionine; and (c) apolypeptide comprising amino acids 1 to 2458 of SEQ ID NO:13.
 9. Theisolated polypeptide of claim 8, wherein the polypeptide comprises twoor more sequential amino acid deletions from one or both of: (a) theCOOH-terminus of the polypeptide; and (b) the NH₂-terminus of thepolypeptide.
 10. A method of identifying a compound that modulates theactivity of the polypeptide of claim 8, the method comprising: (a)determining the activity of the polypeptide of claim 8 in the absence ofa test compound; (b) determining the activity of the polypeptide in thepresence of a test compound; and (c) comparing the activity of thepolypeptide in the presence of the test compound with the activity ofthe polypeptide in the absence of the test compound, wherein a change inthe activity of the polypeptide in the presence of the test compoundrelative to the activity of the polypeptide in the absence of the testcompound indicates that the compound that modulates the activity of thepolypeptide.
 11. An isolated antibody which specifically binds to thepolypeptide of claim
 8. 12. The antibody of claim 11, wherein theantibody is selected from the group consisting of a chimeric antibody, asingle chain antibody, a Fab fragment, and a humanized antibody.
 13. Anisolated nucleic acid molecule comprising a polynucleotide having anucleotide sequence selected from the group consisting of: (a) apolynucleotide encoding an ACC2 polypeptide comprising SEQ ID NO:16; (b)an isolated polynucleotide encoding a rat ACC2 polypeptide comprisingamino acids 2 to 2455 of SEQ ID NO:16 minus the start methionine; (c) anisolated polynucleotide encoding a rat ACC2 polypeptide comprising aminoacids 1 to 2455 of SEQ ID NO:16 including the start codon; (d) the cDNAof ATCC Deposit No. PTA-6054; and (e) a polynucleotide capable ofhybridizing under stringent conditions to the polynucleotide specifiedin (a)-(d), wherein the polynucleotide does not hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence of only A residues or of only T residues.
 14. The isolatednucleic acid molecule of claim 13, wherein the polynucleotide comprisesthe nucleotide sequence of SEQ ID NO:15.
 15. A polynucleotide that iscomplementary to the isolated nucleic acid molecule of claim
 13. 16. Avector comprising the isolated nucleic acid molecule of claim
 13. 17. Ahost cell comprising the vector of claim
 16. 18. The host cell of claim17, wherein the host cell is a mammalian host cell.
 19. A method ofmaking an isolated polypeptide comprising: (a) culturing the recombinanthost cell of claim 17 under conditions such that the polypeptide isexpressed; and (b) recovering the polypeptide.
 20. An isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) a polypeptide comprising SEQ ID NO:16; (b) apolypeptide comprising amino acids 2 to 2455 of SEQ ID NO:16, whereinamino acids 2 to 2455 comprise a polypeptide of SEQ ID NO:16 minus thestart methionine; and (c) a polypeptide comprising amino acids 1 to 2455of SEQ ID NO:16.
 21. The isolated polypeptide of claim 20, wherein thepolypeptide comprises two or more sequential amino acid deletions fromone or both of: (a) the C-terminus of the polypeptide; and (b) theN-terminus of the polypeptide.
 22. A method of identifying a compoundthat modulates the activity of the polypeptide of claim 20, the methodcomprising: (a) determining the activity of the polypeptide of claim 20in the absence of a test compound; (b) determining the activity of thepolypeptide in the presence of a test compound; and (c) comparing theactivity of the polypeptide in the presence of the test compound withthe activity of the polypeptide in the absence of the test compound,wherein a change in the activity of the polypeptide in the presence ofthe test compound relative to the activity of the polypeptide in theabsence of the test compound indicates that the compound that modulatesthe activity of the polypeptide.
 23. An isolated antibody whichspecifically binds to the polypeptide of claim
 20. 24. The antibody ofclaim 23, wherein the antibody is selected from the group consisting ofa chimeric antibody, a single chain antibody, a Fab fragment, and ahumanized antibody.
 25. A method of isolating an ACC polypeptidecomprising: (a) contacting crude lysate derived from a cell or tissueexpressing an ACC polypeptide with an antibody to form a complexcomprising an antibody and ACC; (b) washing the complex with a buffercomprising 0.5 M NaCl; and (c) contacting the complex with an elutingligand.
 26. The method of claim 25, wherein the antibody comprises anIgG antibody.
 27. The method of claim 26, wherein the IgG antibody is ac-Myc-5 IgG antibody and the eluting ligand is a myc peptide.
 28. Themethod of claim 27, wherein the myc peptide comprises the amino acidsequence of SEQ ID NO:17.
 29. The method of claim 26, wherein the IgGantibody is an anti-FLAG IgG antibody.
 30. The method of claim 25,wherein the antibody is bound to a substrate.
 31. The method of claim25, wherein the ACC polypeptide is an ACC1 polypeptide.
 32. The methodof claim 25, wherein the ACC polypeptide is an ACC2 polypeptide.
 33. Themethod of claim 25 wherein the ACC polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:13 and
 16. 34.A polynucleotide capable of inhibiting the expression of an ACC2 genecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NOs:12 and 15 by antisense inhibition.
 35. A method ofinhibiting ACC2 gene expression comprising introducing a polynucleotideof claim 34 into a cell or tissue that expresses an ACC2 gene, therebyinhibiting the expression of the gene in the cell or tissue by antisenseinhibition.
 36. A polynucleotide capable of inhibiting the expression ofan ACC2 gene comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs:12 and 15 by RNA interference.
 37. Amethod of inhibiting ACC2 gene expression comprising introducing apolynucleotide of claim 36 into a cell or tissue that expresses an ACC2gene, thereby inhibiting the expression of the gene in the cell ortissue by RNA interference.
 38. An isolated polypeptide comprising anACC2 polypeptide encoded by the cDNA deposited as ATCC Accession No.PTA-6054.
 39. A method of increasing the activity of a human ACC2polypeptide, the method comprising generating an enhanced ACC2polypeptide comprising (a) a phenylalanine residue at position 254, (b)a glutamine residue at position 346, (c) a threonine residue at position565, (d) an asparagine at position 841, (e) a valine residue at position1103, (f) a cysteine residue at position 1259, (g) an alanine residue atposition 1526, and (h) an isoleucine residue at position 1717, whereinthe human ACC2 polypeptide does not comprise SEQ ID NO:13 and whereinthe enhanced ACC2 polypeptide has an enzymatic activity level that isgreater than the enzymatic activity level of an ACC2 polypeptide thatdoes not contain the indicated residues at the indicated positions. 40.The method of claim 39, wherein the human ACC2 polypeptide sequence isselected from the group consisting of SEQ ID NOs: 2, 4 and 6.